Tao protein kinases and methods of use therefor

ABSTRACT

Compositions and methods are provided for potentiating the activity of the mitogen-activated protein kinase p38. In particular the mitogen-activated protein kinase kinase MEK6, and variants thereof that stimulate phosphorylation of p38 are provided. Such compounds may be used, for example, for therapy of diseases associated with the p38 cascade and to identify antibodies and other agents that inhibit or activate signal transduction via p38.

STATEMENT OF GOVERNMENT INTEREST

The Government owns certain rights in the present invention pursuant to NIH Grant GM53032.

TECHNICAL FIELD

The present invention relates generally to compositions and methods for modulating the activity of the MAP/ERK kinase MEK3 and/or other MEK family members. The invention is more particularly related to TAO proteins, and variants thereof that stimulate phosphorylation and activation of MEK substrates, such as MEK3. The invention is further related to the use of such proteins, for example, to activate a stress-responsive MAP kinase pathway in an organism and to identify antibodies and other agents that inhibit or activate signal transduction via such a pathway.

BACKGROUND OF THE INVENTION

MAP kinase pathways are conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals. Each pathway contains a MAP kinase module, consisting of a MAP kinase or ERK, a MAP/ERK kinase (MEK), and a MEK kinase (MEKK). In higher eucaryotes, activation of MAP kinase pathways has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAP kinase pathways, such as inflammatory diseases, autoimmune diseases and cancer.

Several MAP kinase pathways have been found in S. cerevisiae (Hunter and Plowman, Trends in Biochem. Sci. 22:18-22, 1997), and parallel mammalian pathways have been identified based upon sequences of mammalian ERKs and yeast MAP kinases, KSS1 and FUS3 (Boulton et al., Science 249:64-67, 1990; Courchesne et al., Cell 58: 1107-1119, 1989; Elion et al., Cell 60:649-664, 1990). The best delineated yeast MAP kinase pathway, activated by mating pheromones, is controlled by a receptor-G protein system, includes a Cdc42 small G protein, and requires at least three protein kinases, Ste20p (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad Sci. USA 90:452-456, 1993), Ste11p (Rhodes et al., Genes Dev. 4:1862-1874, 1990), and Ste7p (Teague et al., Proc. Natl. Acad. Sci. USA 83:7371-7375, 1986), upstream of the MAP kinase Fus3p (Elion et al., Cell 60:649-664, 1990).

Ste20p was isolated from S. cerevisiae as a gene whose product functions downstream of the βy subunits of a heterotrimeric G protein but upstream of enzymes in the MAP kinase module (MEKK, MEK, ERK) of the pheromone response pathway (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad. Sci. USA 90:452-456, 1993). Ste11p, the MEKK, may be one of the Ste20p substrates (Wu et al., J. Biol. Chem. 270:15984-15992, 1990); thus, Ste20p-like enzymes may activate MEKKs in mammalian MAP kinase pathways. Ste20p, like its best studied mammalian counterparts, the p21-activated protein kinases (PAKs), is thought to be regulated by binding to Cdc42 through a conserved Cdc42/Rac interactive binding region, or CRIB domain (Burbelo et al., J. Biol. Chem. 270:29071-29074, 1995).

Mammalian relatives of Ste20p are diverse and include the PAK subfamily (PAK1,2,3) and the mixed lineage kinase (MLK) subfamily, including the dual leucine zipper kinase (DLK), germinal center kinase (GCK), and the Nck-interacting kinase, NIK. In the past year, newly identified Ste20p-related kinases include members of the MLK subfamily, SOK-1, Krs-1 and -2, and MUK. MUK was isolated in a screen for MEKK isoforms, but in fact shows more identity to MLK. In transfected cells several of these enzymes, as first shown with GCK, increase the activity of the stress-responsive kinases, particularly SAPK/JNK. In the case of NIK and GCK, they may work by binding to MEKK (Su et al., EMBO J. 16:1279-1290, 1997). However, several of these Ste20p-related enzymes also have MEKK activity. For example, DLK phosphorylates and potently activates MEKs that lie in the stress-responsive cascades.

Further characterization of members of these pathways, and the identification of additional members, is critical for understanding the signal transduction pathways involved and for developing methods for activating or inactivating MEKs and MAP kinase pathways in vivo. Accordingly, there is a need in the art for improved methods for modulating the activity of members of MAP kinase pathways, and for treating diseases associated with such pathways. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods for modulating the activity of MAP/ERK kinases such as MEK3, and stress-responsive MAP kinase pathways. Within certain aspects, the present invention provides TAO polypeptides. Within one such aspect, the polypeptide may comprise an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4, or a variant thereof in which the ability to phosphorylate MEK3 is not substantially diminished. In certain embodiments, such a polypeptide may comprise a sequence that differs from a sequence recited in SEQ ID NO:2 or SEQ ID NO:4 only in conservative substitutions and/or modifications at no more than 10% of the amino acid residues. In certain other embodiments, the polypeptide may be a constitutively active variant.

Within other aspects, the present invention provides polypeptides comprising an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4 modified at no more than 10% of the amino acid residues, such that the polypeptide is rendered constitutively inactive.

Within further aspects, the present invention provides polypeptides capable of phosphorylating MEK3, wherein the polypeptide does not detectably phosphorylate MEK1 or MEK2.

The present invention further provides, within other aspects, isolated polynucleotides encoding polypeptides as described above. Isolated polynucleotides comprising one or more sequences recited in any one of SEQ ID NOs:5-16, or a variant thereof, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3, are also provided. Polypeptides encoded by such polynucleotides are further provided. Recombinant expression vectors comprising any of the above polynucleotides, and host cells transformed or transfected with such expression vectors, are provided within related aspects.

Within other aspects, the present invention provides antisense polynucleotides comprising at least 10 nucleotides complementary to a polynucleotide as described above.

Within further aspects, pharmaceutical compositions are provided, comprising: (a) a polypeptide or polynucleotide as described above; and (b) a physiologically acceptable carrier.

The present invention further provides, within other aspects, methods for phosphorylating a MEK3 polypeptide, comprising contacting a MEK3 polypeptide with a polypeptide according to claim as described above, thereby phosphorylating the MEK3 polypeptide.

Within further aspects, the present invention provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of a stress-responsive MAP kinase pathway.

Within other aspects, methods are provided for phosphorylating a MEK3 polypeptide comprising contacting a MEK3 polypeptide with a polypeptide as described above, and thereby phosphorylating the MEK3 polypeptide.

The present invention further provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of the stress-responsive MAP kinase pathway.

Within further aspects, the present invention provides methods for screening for an agent that modulates signal transduction via a stress-responsive MAP kinase pathway, comprising: (a) contacting a candidate agent with a polypeptide as described above; and (b) subsequently measuring the ability of the polypeptide to modulate the activity of a MEK3 polypeptide, and thereby evaluating the ability of the compound to modulate signal transduction via a stress-responsive MAP kinase pathway.

The present invention further provides, within other aspects, monoclonal antibodies and antigen-binding fragments thereof that specifically bind to a polypeptide as described above. Such monoclonal antibodies or fragments thereof may inhibit the phosphorylation of MEK3 by the polypeptide. Pharmaceutical compositions comprising: (a) an antibody or antigen-binding fragment thereof as described above; and (b) a physiologically acceptable carrier are also provided.

Within other aspects, the present invention provides methods for treating a patient afflicted with a disease associated with a stress-responsive MAP kinase pathway, comprising administering to a patient a compound that modulates the phosphorylation of MEK3. Within certain embodiments, the compound comprises a monoclonal antibody or antigen-binding fragment thereof or a nucleotide sequence. Within such methods, the compound may inhibit phosphorylation of MEK3 and the disease may be inflammation, an autoimmune disease, cancer or a degenerative disease. Alternatively, the compound may enhance the phosphorylation of MEK3 and the disease may be insulin-dependent diabetes or a neurodegenerative disease.

Within other aspects, methods are provided for determining the presence or absence of TAO kinase activity in a sample, comprising evaluating the ability of the sample to phosphorylate a MEK3 polypeptide, and thereby determining the presence or absence of TAO kinase activity in the sample.

Within related aspects, kits are provided for detecting TAO kinase activity in a sample, comprising a MEK3 polypeptide in combination with a suitable buffer.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the nucleotide and predicted amino acid sequence of a representative TAO1 kinase (SEQ ID NOs: 1 and 2).

FIG. 2 presents a comparison of the catalytic domains of TAO1 (residues 1-273 of SEQ ID NO:2), TAO2 (residues 1-273 of SEQ ID NO:4), STE20 (SEQ ID NO:17) and the C. elegans homolog (ceTAO) (SEQ ID NO:18). The catalytic domains were aligned by eye and the conserved amino acids bolded. The domains are indicated with roman numerals.

FIGS. 3A and 3B are Northern blots, showing TAO1 (FIG. 3A) and TAO2 (FIG. 3B) expression is various tissues. Various rat poly-A+ RNAs were probed, as indicated. Equal loading of RNA was verified by hybridizing the blot to an actin probe (not shown).

FIGS. 4A and 4B are Northern blots in which RNAs made from various human brain and spinal cord sections were hybridized to a TAO1-specific probe. Shown below each blot is the result of its hybridization to an actin probe. The lanes are as follows: 1, amygdala, 2, caudate nucleus, 3, corpus callosum, 4, hippocampus, 5, whole brain, 6, substantia nigra, 7, subthalamic nucleus, 8, thalamus, 9, cerebellum, 10, cerebral cortex, 11, medulla, 12, spinal cord, 13, occipital lobe, 14, frontal lobe, 15, temporal lobe, 16, putamen.

FIGS. 5A-5C are immunoblots. In FIG. 5A, human embryonic kidney 293 cells were transiently transfected with either vector or pCMV5TAO1(HA)₃, and 24 hours later lysates were immunoblotted with a monoclonal antibody directed against the HA epitope. TAO1 is indicated by the arrow. In FIG. 5B, the TAO1 proteins purified from Sf9 cells were immunoblotted with an antibody directed against the MRGS(H)₆ epitope. In FIG. 5C, 50 ng of (His)₆ TAO1 was immunoblotted with polyclonal antisera P820 directed against a TAO1 peptide. An equal amount was blotted with the preimmune serum for P820.

FIG. 6 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. Either 50 ng (lanes 1 and 3) or 250 ng (lanes 2 and 4) of (His)₆ TAO1(1-416) was incubated with 50 ng of (His)₆ MEK3 for one hour at 30° in the presence of Mg/ATP, after which a portion of the each reaction was added to a second reaction containing (His)6p38. After a one hour incubation, the reactions were subjected to SDS-PAGE and autoradiography.

FIG. 7 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. Only the second part of the linked assay is shown. The assay was identical to that described in FIG. 6, except that GSTMEK4 was substituted for MEK3, and both (His)₆ p38 and GSTSAPKP were used as MEK4 substrates.

FIG. 8 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. The assay was as described in FIGS. 6 and 7, but was performed with GSTMEK6 and (His)₆ p38 as the MEK6 substrate.

FIG. 9 is a histogram comparing the fold activations of MEKs 1 through 6 by (His)₆ TAO1(1-416).

FIG. 10 is an autoradiogram illustrating TAO1 activation of MEK3 in vivo. Human embryonic kidney 293 cells were transiently transfected with either vector alone, or pCMV5TAO1(HA)₃ and pCMV5mycMEK3, alone and in combination. Immunoprecipitates made with a monoclonal antibody directed against the myc epitope were subjected to in vitro kinase assays with (His)₆ p38 as substrate. Myc-tagged MEK3 expression detected with a polyclonal anti-MEK3 antisera is shown below. In several separate experiments, MEK3 activity in the immunoprecipitates was increased 3 to 4 fold when coexpressed with TAO1.

FIG. 11 is an autoradiogram illustrating the copurification of TAO1 and endogenous MEK3 from Sf9 cells. Either 100 μg of Sf9 whole cell lysate, or 1 μg each of the recombinant TAO1 proteins purified from Sf9 cells was Western blotted with polyclonal antisera directed against MEK3 (top panel) or MEK4 (lower panel). An identical Western blot performed with an antisera against MEK6 did not detect MEK6 protein in either the Sf9 lysate or the TAO1 preparations.

FIG. 12 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:5) with nts. 2341-2754 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 13 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:6) with nts. 964-651 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 14 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:7) with nts. 2792-2423 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIGS. 15A-15B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:8) with nts. 2248-2437 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 15B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:9) with nts. 2437-2501 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 16 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:10) with nts. 2087-2305 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIGS. 17A-17B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:1) with nts. 3228-3312 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 17B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:12) with nts. 3200-3245 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 18 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:13) with nts. 739-854 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIGS. 19A-19B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:14) with nts. 526-643 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 19B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:15) with nts. 187-296 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 20 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:16) with nts. 866-733 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compounds and methods for modulating (i.e., stimulating or inhibiting) the activity of MAP/ERK family members such as the MAP/ERK kinase MEK3. Compounds that activate such MEKs generally stimulate MEK phosphorylation. Such compounds include Ste20p homologs referred to herein as TAO polypeptides (i.e., TAO1, TAO2 or a variant of TAO1 or TAO2 that retains the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein). Alternatively, a compound that activates MEK3 may comprise a polynucleotide that encodes a TAO polypeptide. Within other embodiments, compositions that stimulate MEK3 phosphorylation (thereby activating MEK3) may also, or alternatively, include one or more agents that stimulate TAO polypeptide expression or kinase activity. Such agents include, but are not limited to, stress-inducing agents (e.g., DNA-damaging agents). Additional such agents may be identified by combining a test compound with a TAO polypeptide in vitro and evaluating the effect of the test compound on the kinase activity of the polypeptide using, for example, a representative assay described herein.

Compositions that inhibit the activity of MEKs generally inhibit MEK phosphorylation. Such compositions may include one or more agents that inhibit or block TAO polypeptide activity, such as an antibody that inhibits the kinase activity of a TAO polypeptide, a competing peptide that represents the substrate binding domain of a TAO protein or a phosphorylation motif of the MEK3 substrate, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of a TAO polypeptide, a molecule that inactivates a TAO polypeptide by binding to the polypeptide, a molecule that binds to the TAO substrate and prevents phosphorylation by a TAO polypeptide or a molecule that prevents transfer of phosphoryl groups from the kinase to the substrate. Agents that inhibit TAO polypeptide kinase activity may be identified by combining a test compound with a TAO polypeptide in vitro and evaluating the activity of the TAO polypeptide using a TAO kinase assay.

TAO Polynucleotides

Any polynucleotide that encodes a TAO polypeptide, or a portion or variant thereof as described herein, is encompassed by the present invention. Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a TAO polynucleotide may, but need not, be linked to other molecules and/or support materials.

Native TAO DNA sequences, or portions thereof, may be isolated using any of a variety of hybridization or amplification techniques, which are well known to those of ordinary skill in the art. Within such techniques, probes or primers may be designed based on the TAO sequences provided herein, and may be purchased or synthesized. Libraries from any suitable tissue (e.g., brain) may be screened. An amplified portion or partial cDNA molecule may then be used to isolate a full length gene from a genomic DNA library or from a cDNA library, using well known techniques. Alternatively, a full length gene can be constructed from multiple PCR fragments.

Nucleic acid sequences corresponding to the native rat TAO polypeptides TAO1 and TAO2 are provided in SEQ ID NO:1 and SEQ ID NO:3, respectively. One preferred variant of TAO1 comprises amino acids 1-416 of SEQ ID NO:1. The predicted TAO1 open reading frame encodes a polypeptide of 1001 amino acids with a calculated molecular mass of 134 kD. TAO1 comprises an amino-terminal catalytic domain and an extensive carboxy-terminal region that has several distinguishing features, such as a possible nucleotide binding site and acidic stretch just carboxy-terminal to the catalytic domain, as well as two serine-rich regions. The TAO1 catalytic domain extends 263 amino acids from amino acid 25 to 288 with all 11 of the typical protein kinase subdomains conserved. There are two glutamate residues between TAO1 subdomains II and IV; the second glutamate at amino acid 76 contained in the sequence KEVK is most likely to represent subdomain III (flanks et al., Science 241:42-52, 1988). The features of the TAO1 catalytic domain are most similar to the serine/threonine family of protein kinases; subdomain VIb with the sequence HRDIKAGN (SEQ ID NO:26) suggests that TAO1 is likely to be a serine/threonine protein kinase.

A partial sequence of the closely related gene TAO2 is provided in SEQ ID NOs:3 and 4. TAO2 has a similar arrangement of an amino-terminal kinase domain and a long carboxy-terminus, but differs in that it contains an acidic insert of 17 glutamate residues carboxy-terminal to the catalytic domain, and lacks the putative nucleotide binding site of TAO1.

The rat TAO1 and TAO2 transcripts are highly expressed in brain. Lower levels of TAO1 expression can be observed in heart and lung, with no detectable signal (using a Northern blot analysis as described herein) in skeletal muscle, liver, kidney, testis, epididymus and spleen.

The polynucleotides specifically recited herein, as well as full length polynucleotides comprising such sequences, other portions of full length polynucleotides, and sequences complementary to all or a portion of such full length molecules, are specifically encompassed by the present invention. In addition, TAO homologs from other species are specifically contemplated, and may generally be prepared as described herein for the rat homologs. In particular, within the context of the present invention, EST database sequences derived from retinal mRNAs have been identified that correspond to the human counterpart for TAO1. The sequences of these ESTs are provided in SEQ ID NOs:5-16. It will be readily apparent to those of ordinary skill in the art that a full length, native, human TAO1 polynucleotide may be identified based on such sequences, using for example, standard hybridization or amplification techniques. Such full length TAO1 sequences are contemplated by the present invention, as are polypeptides encoded by such sequences, and variants of the naturally occurring sequences as discussed herein.

Polynucleotide variants of the recited sequences may differ from a native TAO polynucleotide in one or more substitutions, deletions, insertions and/or modifications. Certain variants encode a polypeptide that retains the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein. The effect on the properties of the encoded polypeptide may generally be assessed as described herein. Preferred variants contain nucleotide substitutions, deletions, insertions and/or modifications at no more than 20%, preferably at no more than 10%, of the nucleotide positions. Certain variants are substantially homologous to a native gene, or a potion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a TAO protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5× SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5× SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2× SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

As noted above, the present invention further provides antisense polynucleotides and portions of any of the above sequences. Such polynucleotides may generally be prepared by any method known in the art, including synthesis by, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences that are incorporated into a vector downstream of a suitable RNA polymerase promoter (such as T3, T7 or SP6). Certain portions of a TAO polynucleotide may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may function as a probe (e.g., to detect TAO expression in a sample), and may be labeled by a variety of reporter groups, such as radionuclides, fluorescent dyes and enzymes. Such portions are preferably at least 10 nucleotides in length, and more preferably at least 20 nucleotides in length. Within certain preferred embodiments, a portion for use as a probe comprises a sequence that is unique to a TAO gene. A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression. DNA constructs that can be transcribed into antisense RNA may also be introduced into cells or tissues to facilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Additional initial, terminal and/or intervening DNA sequences that, for example, facilitate construction of readily expressed vectors may also be present. Suitable vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art. Other elements that may be present in a vector will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Vectors as described herein may generally be transfected into a suitable host cell, such as a mammalian cell, by methods well-known in the art Such methods include calcium phosphate precipitation, electroporation and microinjection.

TAO Polypeptides

Polypeptides within the scope of the present invention comprise at least a portion of a TAO protein (e.g., TAO1 or TAO2) or variant thereof, where the portion is immunologically and/or biologically active. Preferred variants retain the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein. A polypeptide may further comprise additional sequences, which may or may not be derived from a native TAO protein. Such sequences may (but need not) possess immunogenic or antigenic properties and/or a biological activity.

A polypeptide "variant," as used herein, is a polypeptide that differs from a native protein in substitutions, insertions, deletions and/or amino acid modifications, such that the immunogenic and/or biological properties of the native protein are not substantially diminished. A variant preferably retains at least 80% sequence identity to a native sequence, more preferably at least 90% identity, and even more preferably at least 95% identity. Within certain preferred embodiments, such variants contain alterations at no more than 10% of the amino acid residues in the native polypeptide, such that the ability of the variant to stimulate MEK3 phosphorylation is not substantially diminished. Guidance in determining which and how many amino acid residues may be substituted, inserted, deleted and/or modified without diminishing immunological and/or biological activity may be found using any of a variety of methods and computer programs known in the art. Properties of a variant may generally be evaluated by assaying the reactivity of the variant with, for example, antibodies as described herein and/or evaluating a biological property characteristic of the native protein.

A polypeptide is "immunologically active," within the context of the present invention if it is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Immunological activity may generally be assessed using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones, which may be prepared using well known techniques. An immunologically active portion of a TAO protein reacts with such antisera and/or T-cells at a level that is not substantially lower than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis.

Similarly, a polypeptide is "biologically active" if the ability to phosphorylate MEK3 and/or other MEKs is not substantially diminished within a representative in vitro assay as described in Example 3. Preferably, the ability of the polypeptide to phosphorylate MEK3 is not substantially diminished. As used herein, the term "not substantially diminished" means retaining an activity that is at least 90% of the activity of a native TAO protein. Appropriate assays designed to evaluate such activity may be designed based on existing assays known in the art, and on the representative assays provided herein.

Preferably, a variant contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes.

In general, modifications may be more readily made in non-critical regions, which are regions of the native sequence that do not substantially change the properties of the TAO protein. Non-critical regions may be identified by modifying the TAO sequence in a particular region and assaying the activity of the resulting variant in a kinase assay, using MEK3, MEK4, MEK6 or another MEK family member as a substrate, as described herein. Modifications may also be made in critical regions of a TAO protein, provided that the resulting variant retains the ability to stimulate MEK3 phosphorylation and/or an immunogenic property of the native protein. Inactive proteins may be created by modifying certain critical regions. One critical region comprises the aspartate 169 residue. Modification of that residue results in a catalytically defective mutant. Another critical region encompasses the lysine 57 residue. The effect of any modification on the ability of the variant to stimulate phosphorylation of MEK3 or other MEKs may generally be evaluated using any assay for TAO kinase activity, such as the representative assays described herein.

Variants of TAO proteins include constitutively active proteins. In general, activation of a TAO protein in vivo requires stimulation by a stimulus such as a stress-inducing agent. Constitutively active variants display the ability to stimulate MEK phosphorylation in the absence of such stimulation. Such variants may be identified using the representative in vivo assays for TAO kinase activity described herein.

TAO proteins may also be modified so as to render the protein constitutively inactive (i.e., unable to phosphorylate MEKs even when stimulated as described above). Such modifications may be identified using the representative assays described herein. Genes encoding proteins modified so as to be constitutively active or inactive may generally be used in replacement therapy for treatment of a variety of disorders, as discussed in more detail below.

Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of a native protein is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini.

The present invention also includes polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNA in bacteria such as E. coli provides non-glycosylated molecules. N-glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-A₁ -Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. Variants having inactivated N-glycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide synthesis and ligation or site-specific mutagenesis techniques, and are within the scope of this invention. Alternatively, N-linked glycosylation sites can be added to a polypeptide.

As noted above, polypeptides may further comprise sequences that are of related to an endogenous TAO protein. For example, an N-terminal signal (or leader) sequence may be present, which co-translationally or post-translationally directs transfer of the polypeptide from its site of synthesis to a site inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader). The polypeptide may also comprise a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His, hemagglutinin, glutathione-S-transferase or FLAG), or to enhance polypeptide stability or binding to a solid support. Protein fusions encompassed by this invention further include, for example, polypeptides conjugated to an immunoglobulin Fc region or a leucine zipper domain. All of the above protein fusions may be prepared by chemical linkage or as fusion proteins.

Also included within the present invention are alleles of a TAO protein. Alleles are alternative forms of a native protein resulting from one or more genetic mutations (which may be amino acid deletions, additions and/or substitutions), resulting in an altered mRNA. Allelic proteins may differ in sequence, but overall structure and function are substantially similar.

TAO polypeptides, variants and portions thereof may generally be prepared from nucleic acid encoding the desired polypeptide using well known techniques. To prepare an endogenous protein, an isolated cDNA may be used. To prepare a variant polypeptide, standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis may be used, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.

In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA sequence that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, baculovirus-infected insect cells and animal cells. Following expression, supernatants from host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Various modified solid phase techniques are also available (e.g., the method of Roberge et al., Science 269:202-204, 1995). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, polypeptides provided herein are isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight. In general, such purification may be achieved using, for example, the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

Antibodies and Fragments Thereof

The present invention further provides antibodies, and antigen-binding fragments thereof, that specifically bind to a TAO polypeptide. As used herein, an antibody, or antigen-binding fragment, is said to "specifically bind" to a TAO polypeptide if it reacts at a detectable level (within, for example, an ELISA) with a TAO polypeptide, and does not react detectably with unrelated proteins. Antibodies may be polyclonal or monoclonal. Preferred antibodies are those antibodies that inhibit or block TAO activity in vivo and within a kinase assay as described herein. Other preferred antibodies (which may be used, for example, in immunokinase assays) are those that immunoprecipitate active TAO1 and/or TAO2.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the polypeptide is initially injected into a suitable animal (e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for a TAO polypeptide may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by, for example, affinity chromatography on protein A bead columns.

Methods and Kits for Detecting TAO Polypeptides and TAO Kinase Activity

The present invention provides methods for detecting the level of TAO1 and/or TAO2 in a sample, as well as for detecting TAO kinase activity in a sample. The level of a TAO polypeptide or polynucleotide may generally be determined using a reagent that binds to the TAO protein, DNA or mRNA. To detect nucleic acid encoding a TAO protein, standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer. Suitable probes and primers may be designed by those of ordinary skill in the art based on the TAO cDNA sequences provided herein. To detect TAO protein, the reagent is typically an antibody, which may be prepared as described herein.

There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a polypeptide in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the antibody may be immobilized on a solid support such that it can bind to and remove the polypeptide from the sample. The bound polypeptide may then be detected using a second antibody that binds to the antibody/peptide complex and contains a detectable reporter group. Alternatively, a competitive assay may be utilized, in which polypeptide that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the level of polypeptide within the sample. Suitable reporter groups for use in these methods include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.

For detecting an active TAO protein in a sample, an immunokinase assay may be employed. Briefly, polyclonal or monoclonal antibodies may be raised against a unique sequence of a TAO protein (such as amino acid residues 296-315, 403-418, 545-563 or 829-848) using standard techniques. A sample to be tested, such as a cellular extract, is incubated with the anti-TAO antibodies to immunoprecipitate a TAO protein, and the immunoprecipitated material is then incubated with a substrate (e.g., MEK3) under suitable conditions for substrate phosphorylation. The level of substrate phosphorylation may generally be determined using any of a variety of assays, as described herein.

TAO kinase assays, for use in evaluating the polypeptide variants and other agents discussed herein, include any assays that evaluate a compound's ability to phosphorylate MEK3 or other MEKs, thereby rendering the MEK active (i.e., capable of phosphorylating in vivo substrates such as p38). MEKs such as MEK3 for use in such methods may be endogenous proteins or variants thereof, may be purified or recombinant, and may be prepared using any of a variety of techniques that will be apparent to those of ordinary skill in the art. For example, cDNA encoding MEK3 may be cloned by PCR amplification from a suitable human cDNA library, using polymerase chain reaction (PCR) and methods well known to those of ordinary skill in the art. MEK3 may be cloned using primers based on the published sequence (Derijard et al., Science 267:682-685, 1995). MEK3 cDNA may then be cloned into a bacterial expression vector and the protein produced in bacteria, such as E. coli, using standard techniques. The bacterial expression vector may, but need not, include DNA encoding an epitope such as glutathione-S transferase protein (GST) such that the recombinant protein contains the epitope at the N- or C-terminus.

A TAO kinase assay may generally be performed as described herein. Briefly, a TAO polypeptide may be incubated with MEK3 and [γ-³² P]ATP in a suitable buffer (such as 50 mM HEPES pH 8, 10 mM MgCl₂, 1 mM DTT, 100 μM ATP) for 60 minutes at 30° C. In general, approximately 50 ng to 1 μg of the polypeptide and 50 ng recombinant MEK3, with 2-7 cpm/fmol [γ-³² P]ATP, is sufficient. Proteins may then be separated by SDS-PAGE on 10% gels and subjected to autoradiography. Incorporation of [³² P]phosphate into MEK3 may be quantitated using techniques well known to those of ordinary skill in the art, such as with a phosphorimager. To evaluate the substrate specificity of polypeptide variants, a kinase assay may generally be performed as described above except that other MEK substrates (i.e., MEK1, 2, 4 or 6) are substituted for the MEK3.

To determine whether MEK3 phosphorylation results in activation, a coupled in vitro kinase assay may be performed using a substrate for MEK3, such as p38, with or without an epitope tag. p38 for use in such an assay may be prepared as described in Han et al., J. Biol. Chem. 271:2886-2891, 1996. Briefly, following phosphorylation of MEK3 as described above, the MEK3 (e.g., 0.1-10 ng) may be incubated with p38 (e.g., 10 μg/ml) and [γ-³² P]ATP in a kinase buffer as described herein. It should be noted that alternative buffers may be used and that buffer composition can vary without significantly altering kinase activity. Reactions may be separated by SDS-PAGE, visualized by autoradiography and quantitated using any of a variety of known techniques. Activated MEK3 will be capable of phosphorylating p38 at a level that is at least 5% above background using such an assay.

The present invention further provides kits for detecting TAO polypeptides and TAO kinase activity. Such kits may be designed for detecting the level of a TAO polypeptide or polynucleotide, or may detect phosphorylation of MEK3 in a direct kinase assay or a coupled kinase assay, in which the level of phosphorylation and/or the kinase activity of MEK3 may be determined. TAO polypeptides and TAO kinase activity may be detected in any of a variety of samples, such as eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms. In general, the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.

A kit for detecting the level of TAO polypeptide or polynucleotide typically contains a reagent that binds to TAO1 and/or TAO2 protein, DNA or RNA. To detect nucleic acid encoding a TAO polypeptide, the reagent may be a nucleic acid probe or a PCR primer. To detect a TAO protein, the reagent is typically an antibody. The kit also contains a reporter group suitable for direct or indirect detection of the reagent (i.e., the reporter group may be covalently bound to the reagent or may be bound to a second molecule, such as Protein A, Protein G, immunoglobulin or lectin, which is itself capable of binding to the reagent). Suitable reporter groups include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Such reporter groups may be used to directly or indirectly detect binding of the reagent to a sample component using standard methods known to those of ordinary skill in the art.

A kit for detecting TAO kinase activity based on measuring the phosphorylation of MEK3 generally comprises MEK3 in combination with a suitable buffer. A kit for detecting TAO kinase activity based on detecting MEK3 activity generally comprises MEK3 in combination with a suitable MEK3 substrate, such as p38. Optionally, the kit may additionally comprise a suitable buffer and/or material for purification of MEK3 after activation and before combination with substrate. Such kits may be employed in direct or coupled kinase assays, which may be performed as described above.

Methods for Identifying Binding Agents and Modulating Agents

The present invention further provides methods for identifying antibodies and other compounds that bind to and/or modulate the activity of a TAO polypeptide. To evaluate the effect of a candidate modulating agent on TAO polypeptide activity, a kinase assay may be performed as described above, except that the candidate modulating agent is added to the incubation mixture. Briefly, the reaction components, which include the composition to be tested and the TAO polypeptide or a polynucleotide encoding the kinase, are incubated under conditions sufficient to allow the components to interact. Subsequently, the effect of composition on kinase activity or on the level of a polynucleotide encoding the kinase is measured. The observed effect on the kinase may be either inhibitory or stimulatory. The increase or decrease in kinase activity can be measured by, for example, adding a radioactive compound such as ³² P-ATP to the mixture of components, and observing radioactive incorporation into MEK3 or other suitable substrate for a TAO polypeptide, to determine whether the compound inhibits or stimulates kinase activity. A polynucleotide encoding the kinase may be inserted into an expression vector and the effect of a composition on transcription of TAO mRNA can be measured, for example, by Northern blot analysis.

Within such assays, the candidate agent may be preincubated with a TAO polypeptide before addition of ATP and substrate. Alternatively, the substrate may be preincubated with the candidate agent before the addition of kinase. Further variations include adding the candidate agent to a mixture of TAO polypeptide and ATP before the addition of substrate, or to a mixture of substrate and ATP before the addition of TAO polypeptide. Any of these assays can further be modified by removing the candidate agent after the initial preincubation step. In general, a suitable amount of antibody or other candidate agent for use in such an assay ranges from about 0.1 μM to about 10 μM. The effect of the agent on TAO kinase activity may then be evaluated by quantitating the incorporation of [³² P]phosphate into MEK3, as described above, and comparing the level of incorporation with that achieved using the TAO polypeptide without the addition of the candidate agent.

TAO kinase activity may also be measured in whole cells transfected with a reporter gene whose expression is dependent upon the activation of MEK3. For example, polynucleotides encoding a TAO polypeptide and a substrate (e.g., MEK3) may be cotransfected into a cell. The substrate may then be immunoprecipitated, and its activity evaluated in an in vitro assay. Alternatively, cells may be transfected with a ATF2-dependent promoter linked to a reporter gene such as luciferase. In such a system, expression of the luciferase gene (which may be readily detected using methods well known to those of ordinary skill in the art) depends upon activation of ATF2 by p38, which may be achieved by the stimulation of MEK3 with a TAO polypeptide. Candidate modulating agents may be added to the system, as described below, to evaluate their effect on TAO polypeptide activity.

Alternatively, a whole cell system may employ only the transactivation domain of ATF2 fused to a suitable DNA binding domain, such as GHF-1 or GAL4. The reporter system may then comprise the GH-luciferase or GAL4-luciferase plasmid. Candidate TAO protein modulating agents may then be added to the system to evaluate their effect on ATF2-specific gene activation.

In other aspects of the subject invention, methods for using the above polypeptides to phosphorylate and activate MEK3, peptide derivatives thereof or other MEK family members are provided. MEK substrate for use in such methods may be prepared as described above. In one embodiment, MEK3 may be phosphorylated in vitro by incubation with a TAO polypeptide and ATP in a suitable buffer as described above. In general, the amounts of the reaction components may range from about 0.1 μg to about 10 μg of TAO polypeptide, from about 0.1 μg to about 10 μg of recombinant MEK3, and from about 100 nM to about 1 mM (preferably about 100 pmol-30 nmol) of ATP. Phosphorylated proteins may then be purified by binding to GSH-sepharose and washing. The extent of MEK3 phosphorylation may generally be monitored by adding [γ-³² P]ATP to a test aliquot, and evaluating the level of MEK3 phosphorylation as described above. The activity of the phosphorylated MEK3 may be evaluated using a coupled in vitro kinase assay, as described above.

Once activated in vitro, MEK3 may be used, for example, to identify agents that inhibit the kinase activity of MEK3. Such inhibitory agents, which may be antibodies or drugs, may be identified using the coupled assay described above. Briefly, a candidate agent may be included in the mixture of MEK3 and p38, with or without pre-incubation with one or more components of the mixture, as described above. In general, a suitable amount of antibody or other agent for use in such an assay ranges from about 0.1 μM to about 10 μM. The effect of the agent on MEK3 kinase activity may then be evaluated by quantitating the incorporation of [³² P]phosphate into p38, as described above, and comparing the level of incorporation with that achieved using activated MEK3 without the addition of a candidate agent.

Within other aspects, TAO polypeptides may be used to identify one or more native upstream kinases (i.e., kinases that phosphorylate and activate TAO1 and/or TAO2 in vivo, or other signaling molecules that regulate TAO activity). TAO polypeptides may be used in a yeast two-hybrid system to identify interacting proteins. Alternatively, an expression library may be screened to identify cDNAs that encode proteins which phosphorylate a TAO polypeptide. Other methods for identifying such upstream kinases may also be employed, and will be apparent to those of ordinary skill in the art.

Pharmaceutical Compositions

For administration to a patient, one or more polypeptides, polynucleotides, antibodies and/or modulating agents are generally formulated as a pharmaceutical composition, which may be a sterile aqueous or non-aqueous solution, suspension or emulsion, and which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in a pharmaceutical composition. Representative carriers include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials. Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, antimicrobial compounds, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), inert gases and/or preservatives. Compositions of the present invention may also be formulated as a lyophilizate. Pharmaceutical compositions may also contain other compounds, which may be biologically active or inactive.

The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or modulating agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Certain pharmaceutical compositions contain DNA encoding a polypeptide, antibody fragment or other modulating agent as described above (such that a TAO polypeptide, a variant thereof or a modulating agent is generated in situ) or an antisense polynucleotide. In such pharmaceutical compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993.

Various viral vectors that can be used to introduce a nucleic acid sequence into the targeted patient's cells include, but are not limited to, vaccinia or other pox virus, herpes virus, retrovirus, or adenovirus. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or. selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific). For example, retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation. Such helper cell lines include (but are not limited to) Ψ2, PA317 and PA12. A retroviral vector introduced into such cells can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for TAO polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

Routes and frequency of administration, as well as polypeptide, modulating agent or nucleic acid doses, will vary from patient to patient. In general, the pharmaceutical compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily. A suitable dose is an amount of polypeptide or DNA that is sufficient to show improvement in the symptoms of a patient afflicted with a disease associated with a stress-responsive MAP kinase pathway. Such improvement may be detected based on a determination of relevant cytokine levels (e.g., IL-2, IL-8), by monitoring inflammatory responses (e.g., edema, transplant rejection, hypersensitivity) or through an improvement in clinical symptoms associated with the disease. In general, the amount of polypeptide present in a dose, or produced in situ by DNA present in a dose, ranges from about 1 μg to about 250 μg per kg of host, typically from about 1 μg to about 60 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal.

Therapeutic Applications

The above polypeptides, polynucleotides and/or modulating agents may be used to phosphorylate (and thereby activate) MEK3, or to inhibit such phosphorylation, in a patient. As used herein, a "patient" may be any mammal, including a human, and may be afflicted with a disease associated with a stress-responsive MAP kinase pathway, or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Diseases associated with a stress-responsive MAP kinase pathway include any disorder which is etiologically linked to a TAO protein kinase activity, including immune-related diseases (e.g., inflammatory diseases, autoimmune diseases, malignant cytokine production or endotoxic shock), cell growth-related diseases (e.g., cancer, metabolic diseases, abnormal cell growth and proliferation or cell cycle abnormalities) and cell regeneration-related diseases (e.g. cancer, degenerative diseases, trauma, environmental stress by heat, UV or chemicals or abnormalities in development and differentiation). Immunological-related cell proliferative diseases such as osteoarthritis, ischemia, reperfusion injury, trauma, certain cancers and viral disorders, and autoimmune diseases such as rheumatoid arthritis, diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, and other acute phase responses may also be treated.

Treatment includes administration of a composition or compound which modulates the kinase activity of TAO1 and/or TAO2. Such modulation includes the suppression of TAO expression and/or activity when it is over-expressed, or augmentation of TAO expression and/or activity when it is under-expressed. Modulation may also include the suppression of phosphorylation of MEK3 or related kinases.

As noted above, antibodies, polynucleotides and other agents having a desired effect on TAO expression and/or activity may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the activation of MEK3 in vivo. For example, an agent that decreases TAO activity in vivo may be administered to prevent or treat inflammation, autoimmune diseases, cancer or degenerative diseases. In particular, such agents may be used to prevent or treat insulin-resistant diabetes, metabolic disorders and neurodegenerative diseases. In general, for administration to a patient, an antibody or other agent is formulated as a pharmaceutical composition as described above. A suitable dose of such an agent is an amount sufficient to show benefit in the patient based on the criteria noted above.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1 Cloning and Sequencing cDNA Encoding TAO1 and TAO2

This Example illustrates the cloning of cDNA molecules encoding the rat Ste20p-related protein kinases TAO1 and TAO2, and the identification of the human TAO1 homolog.

First-strand cDNA from adult rat brain was used as the template in the first round of PCR with degenerate oligonucleotide primers derived from the Ste20p sequence, 5'-GACGCTGGATCCAA(AG)AT(ACT)GGICA(AG)GGIGC-3' (SEQ ID NO:19) and 5'-GGIGTICC(AG)TTIGTIGCIAT-3' (SEQ ID NO:20). A portion of the product of this reaction was used as the template in a second round of PCR with nested primers, also derived from the Ste20p sequence, 5'-AA(AG)GA(AG)CAIATI(CA)TIAA(CT)GA(AG)AT-3' (SEQ ID NO:21) and 5'-GACGCTGAATTCAC(CT)TCIGGIGCCATCCA-3' (SEQ ID NO:22). The resulting 420 base product was labeled with [α-³² P]dCTP by random-priming, and used to probe approximately 1×10⁶ plaques of an oligo(dT) and random-primed λZAP library generated from adult rat forebrain RNA. In excess of 100 positive clones were obtained; of those sequenced, all contained regions of overlap with the original PCR product. A full length TAO1 sequence was assembled from two overlapping cDNAs, using the Sacd site at nucleotide 50 to insert a fragment of TAO1 cDNA including nucleotides 50 to 3003. The full length TAO1 sequence is shown in FIG. 1 and SEQ ID NO:1.

The TAO1 open reading frame encodes 1001 amino acids, with a calculated molecular mass of 134 kDa. The presumed initiator codon begins at base 121 and is preceded by an in-frame stop codon at base 106. The longest 5' UTR obtained was 600 nucleotides in length, and the longest 3' UTR was 1200 nucleotides. None of the clones analyzed contained a poly-A track.

As is the case with most protein kinases, TAO1 can be divided into regions based on amino acid sequence comparison to other protein kinases. TAO1 is composed of an amino-terminal catalytic domain and an extensive carboxy-terminal region that has several distinguishing features, such as a possible nucleotide binding site and acidic stretch just carboxy-terminal to the catalytic domain, as well as two serine-rich regions. TAO1 does not appear to contain the leucine zipper motifs found in the MLK subfamily of kinases.

The TAO1 catalytic domain extends 263 amino acids from amino acid 25 to 288 with all 11 of the typical protein kinase subdomains conserved. There are two glutamate residues between TAO1 subdomains II and IV; the second glutamate at amino acid 76 contained in the sequence KEVK is most likely to represent subdomain III (Hanks et al., Science 241:42-52, 1988). The features of the TAO1 catalytic domain are most similar to the serine/threonine family of protein kinases; subdomain VIb with the sequence HRDIKAGN suggests that TAO1 is likely to be a serine/threonine protein kinase.

When using FASTA (GCG, Wisconsin Package) to align TAO1 with sequences from the databases, the TAO1 catalytic domain shows the highest degree of identity to a C. elegans putative serine/threonine protein kinase (accession number U32275), to which it has 63% identity and 79% similarity. That sequence appears to represent the C. elegans homolog of TAO1, and is shown as ceTAO in FIG. 2. The TAO1 catalytic domain is 39% identical to Ste20p and 40% identical to the catalytic domains of the p21-activated kinases PAK1 and PAK2. The catalytic domain of TAO1 is only 31% identical to the mixed lineage kinase MLK1, and 33% identical to dual leucine zipper-bearing kinase (DLK), also known as MLK2. Thus, TAO1 appears to be more closely related to the STE20-like kinases than to the MLK family. TAO1 is also related to germinal center kinase (GCK) and mammalian Ste20-like kinase 1 (MST1), with 42% and 45% identity respectively in the catalytic domains. The TAO1 sequence has similarity with that of the MEK kinase MEKK1. Although the overall identity between the catalytic domains of TAO1 and MEKK is only 33%, the identity of the carboxy-terminal half of their catalytic domains is higher (42%).

In the process of screening the cDNA library for clones near the 5' end of TAO1, multiple clones representing a second closely related gene (TAO2) were identified. The TAO2 sequence is provided in SEQ ID NO:3, with the predicted amino acid sequence shown in SEQ ID NO:4 and FIG. 2. TAO2 is highly related to TAO1, and has a similar arrangement of an amino-terminal kinase domain and a long carboxy-terminus, but differs in that it contains an acidic insert of 17 glutamate residues carboxy-terminal to the catalytic domain, and lacks the putative nucleotide binding site of TAO1.

Sequences from EST databases derived from retinal mRNAs revealed the human counterpart for TAO1. The EST sequences identified are provided in SEQ ID NOs:5-16, and the alignments of these sequences with the rat TAO1 sequence are provided in FIGS. 12-20.

The Fasta program was used to compare the percent amino acid identities of several protein kinase catalytic domains, and the results are presented in Table 1, below.

                                      TABLE 1                                      __________________________________________________________________________          TAO1                                                                                                       TAO2 90 TAO2                                    ceTAO 65 61 ceTAO                                                              STE20d 40 39 37 STE20                                                          GCK 43 42 35 40 GCK                                                            MLK1 32 30 27 30 29 MLK1                                                       MST1 47 43 42 42 47 28 MST1                                                    MEKK1 34 33 27 30 30 30 29                                                   __________________________________________________________________________

To assess the expression of TAO1 in transfected cells, full-length, HA-tagged TAO1 cDNA was transfected into human embryonic kidney 293 cells. A protein of approximately 140 kDa could be detected by Western blotting with an antibody directed against the HA epitope (FIG. 5A). The observed molecular mass of the protein is in good agreement with the mass predicted from the cDNA sequence.

Example 2 In vivo Expression of TAO1 and TAO2

This Example illustrates the expression of TAO1 and TAO2 in a variety of adult rat and human tissues, as determined by Northern blot analysis.

Total RNA isolated from various adult male rat tissues was selected for poly-A+ RNA with oligo(dT)cellulose (Collaborative Biomedical Products) according to the manufacturers protocols, and 5 μg of each RNA was subjected to Northern analysis. The PCR-generated 420 base fragment derived from the catalytic domain of TAO1 (described above) was labeled with [α-³² P]dCTP by random-priming and used to probe the Northern blot. Hybridization was at 42° C., followed by washing at 55° C. in 0.2%SSC/0.1%SDS. Integrity of the mRNA was confirmed by hybridization to an actin probe. The TAO1 probe hybridized predominately to an mRNA species of approximately 12 kb, and less strongly to another of approximately 10 kb (FIG. 3A). Of the rat tissues examined, brain clearly showed the strongest hybridization signal. On prolonged exposure, heart and lung revealed weak hybridization signals, while in skeletal muscle, liver, kidney, testis, epididymus, and spleen no signal was detected.

To assess the expression pattern of TAO2, the rat tissue Northern blot was stored until the hybridization signal for TAO1 was not seen on a two week exposure at -80° C. A fragment from the catalytic domain of TAO2 was labeled with [α-³² P]dCTP by random priming, and used to probe the Northern under the same hybridization and washing conditions described above for TAO1.

When the same rat tissue Northern blot was probed with a fragment of the catalytic domain of TAO2, the strongest hybridization signal was also seen in brain. The size of the transcript hybridizing to the TAO2 probe was smaller than that seen for TAO1, at 5 kb (FIG. 3B).

A probe from the non-catalytic carboxy-terminus of TAO1 (corresponding to nucleotides 1555 to 2632 of TAO1 (see FIG. 1)) was used for all additional Northern analyses because it is less likely to hybridize to TAO2 mRNA. This probe from the carboxy-terminus of TAO1 was used to assess the expression pattern in sections of human brain (Clontech). Hybridizations were performed at 68° C. in Clontech ExpressHyb buffer, and washed at 55° C. as per the manufacturer's instructions.

The strongest hybridization signals were seen in amygdala, corpus callosum, hippocampus, and substantia nigra, and each of these was stronger than that seen in whole brain (FIG. 4A). Weaker signals were seen in caudate nucleus, subthalamic nucleus and thalamus. A second human brain Northern hybridized to the same probe showed strong hybridization signals in cerebellum, putamen and occipital, frontal and temporal lobes, but much weaker signals in cerebral cortex, medulla and spinal cord (FIG. 4B).

Example 3 Kinase Activity and Substrate Specificity of TAO1

This Example illustrates the kinase activity and substrate specificity of TAO1, in in vitro and in vivo assays.

To determine whether TAO1 is active as a protein kinase, two constructs were employed. pCMV5TAO1-HA₃ and pCMV5TAO1(1-416)-HA, were generated by cloning the cDNAs encoding these TAO1 polypeptides into the pCMV5 mammalian expression vector. Oligonucleotide primers were used with TAO1 cDNA as template to amplify a 1247 base pair DNA product encoding amino acids 1 to 416. This fragment contains all 11 of the kinase subdomains (with the initial methionine deleted). The resulting constructs were transfected into human embryonic kidney 293 cells, and the recombinant, tagged proteins immunoprecipitated with an antibody directed against the HA epitope.

In vitro kinase assays were generally performed as follows. Kinase assays contained: 50 mM Hepes, pH 8, 10 mM MgCl₂, 1 mM DTT, 100 μM ATP, [γ-³² P]ATP (at a final concentration of 2-7 cpm/fmol), and unless otherwise noted, reactions were incubated at 30° C. for 60 minutes in a 30 μl volume. Protein kinase substrates such as myelin basic protein were added at a final concentration of 0.5 mg/ml. Reactions were halted by the addition of 10μl 5× Laemmli buffer, followed by boiling, and 20 μl were analyzed by SDS-PAGE and autoradiography. For linked kinase assays, 50-250 ng of recombinant TAO1 protein was incubated with 50 ng of each of the bacterially expressed MEK proteins in a 30 μl reaction volume for 60 minutes at 30° C., and then 5 μl of this reaction was added to a second reaction mix containing bacterially expressed (His)₆ p38 or GST-SAPKβ at a final concentration of 10 μg/ml. Recombinant MEK proteins were kindly provided by Andrei Khokhlatchev and Megan Robinson, and may be prepared as described by Robinson et al., J. Biol. Chem. 271:29734-29739, 1996 and references cited therein. Within such assays, both TAO1(1-416) and full-length TAO1 were able to phosphorylate MBP in immune complex kinase reactions.

To quantitate the activity of more highly purified TAO1, TAO1(1-416), full-length TAO1 and full-length TAO1(D169A) were expressed with an amino-terminal hexa-histidine tag in Sf9 cells. TAO1(D169A) is a catalytically defective TAO1 mutant, which was created by changing aspartic acid 169 to an alanine (D169A) with PCR, and cloning the resulting construct into the pCMV5 mammalian expression vector. These constructs were prepared with either a single hemagglutinin (HA) epitope tag at the amino-terminus, a triple HA epitope tag at the carboxy-terminus, or a myc epitope tag at the amino-terminus.

The recombinant, hexa-histidine tagged TAO1, TAO1(1-416), and TAO1(D169A) were expressed in Spodoptera frugiperda (Sf9) cells. Cells were lysed by douncing in 50 mM sodium phosphate, pH 8.5, 1 mM DTT, 1 mM PMSF, and 1 mg/ml each leupeptin, pepstatin A, and aprotinin. After centrifugation at 30,000×g for 30 minutes, the supernatant was applied to a Ni²⁺ -NTA agarose (Qiagen) column pre-equilibrated with the same buffer. The column was then washed with 50 column volumes of buffer, and eluted with a 20 ml gradient of 0 to 250 mM imidazole, all in the above buffer. Fractions containing recombinant TAO1 proteins were detected in fractions by Western blotting with an antibody to the MRGS(H)₆ epitope (Qiagen), and appropriate fractions were pooled and dialyzed to remove the imidazole.

(His)₆ TAO1(1-416) expressed as a single 57 kDa band (FIG. 5B). Both the (His)₆ TAO1 and (His)₆ TAO1(D169A) recombinant proteins migrated as 140 kDa bands, although the D169A mutant appears to be more subject to degradation. (His)₆ TAO1(1-416) phosphorylates MBP with a specific activity of 1 μmolmin⁻¹ mg¹ - in the presence of 1 mM ATP. Full-length (His)₆ TAO1 exhibits MBP phosphorylating activity that is comparable to the 1-416 truncation mutant, while the activity of TAO1(D169A) is reduced to 90% of that of the wild-type protein. (His)₆ TAO1(1-416) was also able to phosphorylate α-casein, histone 1, and histone 7.

To determine whether TAO1 activates one or more of the known MEKs, (His)₆ TAO1(1-416) was incubated with bacterially produced MEK for one hour in the presence of Mg²⁺ and [γ⁻³² P]ATP. A portion of this reaction was then transferred to a similar reaction containing the appropriate bacterially expressed MEK substrate, (His)₆ ERK2K52R for MEK1 and MEK2, (His)₆ p38 for MEK3 and MEK6, and (His)₆ p38 and GST-SAPKβ for MEK4. After a one hour incubation, the phosphoproteins were separated by SDS-PAGE. Autoradiography revealed that (His)₆ TAO1(1-416) phosphorylated and activated (His)₆ MEK3, and enhanced the ability of MEK3 to phosphorylate p38 by approximately 100-fold (FIG. 6).

(His)₆ TAO1(1-416) activated GST-MEK4 5-fold toward (His)₆ p38, and 150-fold towards GST-SAPKβ (FIG. 7). The difference in fold activation seen for MEK4 towards the two substrates probably reflects the difference in basal kinase activity of MEK4 towards p38 and SAPKβ in vitro. TAO1 also increased the ability of GST-MEK6 to phosphorylate (His)₆ p38, by 5-fold (FIG. 8). Recombinant GST-MEK5 was not phosphorylated by (His)₆ TAO1(1-416).

Recombinant (His)₆ TAO1 and (His)₆ TAO1(D169A) were also examined for their ability to activate the same MEK proteins. (His)₆ TAO1 showed a reduced ability to activate MEK3 as compared to that of the carboxy-terminal truncation mutant (His)₆ TAO1(1-416). In multiple experiments, the full-length TAO protein displayed from 0 to 30% of the MEK3 activating ability of (His)₆ TAO1(1-416), and (His)₆ TAO1(D169A) was unable to activate any of the MEK proteins above basal activities.

The degree of activation of each of the MEK proteins by (His)₆ TAO1(1-416) in vitro is comparable to that seen by a bacterially produced amino-terminal truncation of MEKK1 (Xu et al., Proc. Natl. Acad. Sci. USA 92:6808-6812, 1995; Robinson et al., J. Biol. Chem. 271:29734-29739, 1996). To distinguish the MEK-activating ability of TAO1 from that of MEKK, the ability of (His)₆ TAO1(1-416) to activate MEK1 and MEK2 was assessed. As shown in FIG. 9, (His)₆ TAO1(1-416) was completely unable to increase the activity of MEK1 or MEK2 towards the substrate (His)₆ ERK2 under the same conditions that TAO1 activates MEK3, MEK4, and MEK6. Thus, while TAO1 displays MEKK-like activity in its ability to activate various MEKs, TAO1 is differentiated from MEKK by its inability to recognize MEK1 and MEK2. FIG. 9 shows the fold activation of the various MEKs by TAO1.

To assess the ability of TAO1 to activate the various MEKs in vivo, full-length HA-tagged TAO1 was co-transfected into 293 cells with myc-tagged MEK3, or myc-tagged TAO1 was co-transfected with HA-tagged MEK4 or HA-tagged MEK6. The pCMV5myc-MEK3 construct was generated by inserting the MEK3 coding sequence (provided by K. L. Guan, University of Michigan, which may be prepared as described by Robinson et al., J. Biol. Chem. 271:29734-29739, 1996) into the pCMV5Myc vector, such that the Myc epitope is at the amino-terminus of MEK3. The MEKs were then immunoprecipitated and added to immune complex kinase assays with the appropriate substrate and Mg²⁺ /ATP. In multiple experiments, myc-tagged MEK3 showed a 3-fold higher activity toward p38 when immunoprecipitated from 293 cells co-expressing TAO than from cells not transfected with TAO (FIG. 10). In contrast, TAO was not able to increase the activity of immunoprecipitated HA-tagged MEK4 towards GST-SAPKβ, or that of HA-tagged MEK6 toward p38.

In transfected cells, TAO1 activates MEK3 3-fold, but neither MEK4 nor MEK6. The selectivity in transfected cells may arise from the ability of TAO1 to bind MEK3. The endogenous MEK3 from Sf9 cells copurifies with recombinant TAO1 expressed in the cells. These findings suggest that TAO1 may be an important regulator of the p38 pathway.

To determine which MEK3 residues are phosphorylated by TAO, an in vitro kinase reaction was performed with (His)₆ TAO1(1-416) and (His)₆ MEK3; the 57 kDa band corresponding to TAO1 and the 30 kDa band corresponding to MEK3 were excised and treated as described. Phosphoproteins were separated by SDS-PAGE, transferred to Immobilon-P membrane (Millipore) electrophoretically, and visualized by autoradiography. Bands of interest were excised and hydrolyzed in 6M HCl for 60 minutes at 110° C. The hydrolysate was dried under vacuum, and resuspended in a 2.2% formic acid, 12% acetic acid solution at an activity of 2000 cpm/μl. Then 1 μl of each sample was mixed with 1 μg each of the three phosphoamino acid standards, and spotted onto cellulose thin-layer chromatography plates. Electrophoresis was performed in 0.5% pyridine, 5% acetic acid at 1200 volts for 60 minutes. After air drying the plates, the standards were visualized with 0.25% ninhydrin in acetone. Autoradiography revealed only phosphoserine and phosphothreonine in both (His)₆ TAO1 (1-416) and (His)₆ MEK3 (FIG. 11).

Example 4 Co-Purification of MEK3 and TAO1

This Example shows that TAO1 and MEK3 co-purify.

Although the ability of (His)₆ TAO1 to activate MEK3 was always reduced in comparison with that of (His)₆ TAO1(1-416), several assays showed that the ability of (His)₆ TAO1 to lead to an increase in the phosphorylation of p38 in the linked kinase assays was partly independent of the addition of MEK. (His)₆ TAO1(1-416) does not phosphorylate p38. Therefore, Western analyses were performed to determine if one or more MEKs might be present in the TAO1 preparations purified from Sf9 cells.

(His)₆ TAO1, (His)₆ TAO1(1-416), and (His)₆ TAO1(D169A) were subjected to Western analysis with antisera specific to MEK3, MEK4, and MEK6. Four different polyclonal antisera were raised to these three TAO1 peptides in rabbits. The peptide TKDAVRELDNLQYRKMKKLL (SEQ ID NO:23) corresponding to the amino acids 296 to 315 yielded antisera P820. The peptide KKELNSFLESQKREYKLRK (SEQ ID NO:24) of amino acids 545 to 563 yielded the antiserum R562. Finally, the peptide RELRELEQRVSLRRALLEQK (SEQ ID NO:25) of amino acids 829 to 848 resulted in the antisera R564 and R565. These peptides were conjugated to Limulus hemocyanin (Boulton and Cobb, Cell. Regul. 2:357-371, 1991) and dialyzed into phosphate-buffered saline. A total of five boosts were performed, after which the rabbits were exsanguinated and the serum collected. The antisera were screened for reactivity by Western blotting of recombinant TAO1 expressed in Sf9 cells. Five antisera were found to consistently recognize the recombinant TAO1 protein in Western blots. Free peptide was able to block the specific recognition of TAO1 protein by the antisera. None of the five antisera detected the presence of TAO1 in lysates of 293, NIH3T3, NG-108, or COS cells.

For immunoblot analysis, either 50 ng of recombinant TAO1 protein or 100 μg of cell lysate was subjected to SDS-PAGE, then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat powdered milk in TBST (20 mM Tris, pH 8, 500 mM NaC1, 0.05% Tween 20) for one hour, then incubated with the polyclonal antisera at 1:500 dilution in TBST plus 0.25% milk for one hour. After three washes with TBST, the membranes were incubated with a 1:2500 dilution of horseradish peroxidase-conjugated goat-anti-rabbit IgG in TBST plus 0.25% milk for one hour. Membranes were washed again in TBST then visualized with the ECL system (Amersham).

MEK3 was clearly seen in the (His)₆ TAO1 preparation, and to a lesser extent in the (His)₆ TAO1(D169A) preparation (FIG. 11). MEK4 was detected in the Sf9 cell lysates, but not in the TAO1 preparations, while MEK6 was detected in neither.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 26                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3312 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 121..3123                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                            - - TCTGCAGTAT GGTAGATTAT TATTTATGCA TTTATGCCAG TGTGGCTTCA TT -             #CATACAGA     60                                                                  - - TGAACCAAGC TTTGGGATAG CAGTATAAAA TTAGAATCAG ACAGCTGACT GC -             #TCAGCAGG    120                                                                  - - ATG CCA TCA ACT AAC AGA GCA GGC AGT CTA AA - #G GAC CCT GAA ATC         GCA      168                                                                     Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Ly - #s Asp Pro Glu Ile Ala             1               5 - #                 10 - #                 15               - - GAG CTC TTC TTC AAA GAA GAT CCG GAA AAA CT - #C TTC ACA GAT CTC AGA           216                                                                        Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Le - #u Phe Thr Asp Leu Arg                         20     - #             25     - #             30                   - - GAA ATC GGC CAT GGG AGC TTT GGA GCA GTT TA - #T TTT GCA CGA GAT GTG           264                                                                        Glu Ile Gly His Gly Ser Phe Gly Ala Val Ty - #r Phe Ala Arg Asp Val                     35         - #         40         - #         45                       - - CGT ACT AAT GAA GTG GTG GCC ATC AAG AAA AT - #G TCT TAT AGT GGA AAG           312                                                                        Arg Thr Asn Glu Val Val Ala Ile Lys Lys Me - #t Ser Tyr Ser Gly Lys                 50             - #     55             - #     60                           - - CAG TCT ACT GAG AAA TGG CAG GAT ATT ATT AA - #G GAA GTC AAG TTT CTA           360                                                                        Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Ly - #s Glu Val Lys Phe Leu             65                 - # 70                 - # 75                 - # 80        - - CAA AGA ATA AAA CAT CCC AAC AGT ATA GAA TA - #C AAA GGC TGC TAT TTA           408                                                                        Gln Arg Ile Lys His Pro Asn Ser Ile Glu Ty - #r Lys Gly Cys Tyr Leu                             85 - #                 90 - #                 95               - - CGT GAA CAC ACA GCA TGG CTT GTA ATG GAA TA - #T TGT TTA GGA TCT GCT           456                                                                        Arg Glu His Thr Ala Trp Leu Val Met Glu Ty - #r Cys Leu Gly Ser Ala                        100      - #           105      - #           110                   - - TCG GAT TTA CTA GAA GTT CAT AAA AAG CCA TT - #A CAA GAA GTG GAA ATA           504                                                                        Ser Asp Leu Leu Glu Val His Lys Lys Pro Le - #u Gln Glu Val Glu Ile                    115          - #       120          - #       125                       - - GCA GCA ATT ACA CAT GGT GCT CTC CAG GGA TT - #A GCT TAT TTA CAT TCT           552                                                                        Ala Ala Ile Thr His Gly Ala Leu Gln Gly Le - #u Ala Tyr Leu His Ser                130              - #   135              - #   140                           - - CAT ACC ATG ATC CAT AGA GAT ATC AAA GCA GG - #A AAT ATC CTT CTG ACA           600                                                                        His Thr Met Ile His Arg Asp Ile Lys Ala Gl - #y Asn Ile Leu Leu Thr            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - GAA CCA GGC CAA GTG AAA CTT GCT GAC TTT GG - #A TCT GCT TCC ATG         GCC      648                                                                     Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gl - #y Ser Ala Ser Met Ala                           165  - #               170  - #               175               - - TCC CCT GCC AAT TCT TTT GTG GGA ACA CCA TA - #T TGG ATG GCC CCA GAA           696                                                                        Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Ty - #r Trp Met Ala Pro Glu                        180      - #           185      - #           190                   - - GTA ATT TTA GCC ATG GAT GAA GGA CAA TAT GA - #T GGC AAA GTT GAT GTA           744                                                                        Val Ile Leu Ala Met Asp Glu Gly Gln Tyr As - #p Gly Lys Val Asp Val                    195          - #       200          - #       205                       - - TGG TCT CTT GGA ATA ACA TGT ATT GAA TTA GC - #C GAG AGG AAG CCT CCT           792                                                                        Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Al - #a Glu Arg Lys Pro Pro                210              - #   215              - #   220                           - - TTA TTT AAT ATG AAT GCA ATG AGT GCC TTA TA - #T CAC ATA GCC CAA AAT           840                                                                        Leu Phe Asn Met Asn Ala Met Ser Ala Leu Ty - #r His Ile Ala Gln Asn            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - GAA TCC CCT ACA CTA CAG TCT AAT GAA TGG TC - #T GAT TAT TTT CGA         AAC      888                                                                     Glu Ser Pro Thr Leu Gln Ser Asn Glu Trp Se - #r Asp Tyr Phe Arg Asn                           245  - #               250  - #               255               - - TTT GTA GAT TCT TGC CTC CAG AAA ATC CCT CA - #A GAT CGC CCT ACA TCA           936                                                                        Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gl - #n Asp Arg Pro Thr Ser                        260      - #           265      - #           270                   - - GAG GAA CTT TTA AAG CAC ATG TTT GTT CTT CG - #A GAG CGC CCT GAA ACA           984                                                                        Glu Glu Leu Leu Lys His Met Phe Val Leu Ar - #g Glu Arg Pro Glu Thr                    275          - #       280          - #       285                       - - GTG TTA ATA GAT CTT ATT CAA AGG ACA AAG GA - #T GCA GTA AGA GAG CTG          1032                                                                        Val Leu Ile Asp Leu Ile Gln Arg Thr Lys As - #p Ala Val Arg Glu Leu                290              - #   295              - #   300                           - - GAC AAT CTA CAA TAT CGA AAG ATG AAG AAA CT - #C CTT TTC CAG GAG GCA          1080                                                                        Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Le - #u Leu Phe Gln Glu Ala            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - CAT AAT GGA CCA GCA GTA GAA GCA CAG GAA GA - #A GAG GAG GAG CAA         GAT     1128                                                                     His Asn Gly Pro Ala Val Glu Ala Gln Glu Gl - #u Glu Glu Glu Gln Asp                           325  - #               330  - #               335               - - CAT GGT GGT GGC CGG ACA GGA ACA GTA AAT AG - #T GTT GGA AGC AAT CAG          1176                                                                        His Gly Gly Gly Arg Thr Gly Thr Val Asn Se - #r Val Gly Ser Asn Gln                        340      - #           345      - #           350                   - - TCT ATC CCC AGT ATG TCT ATC AGT GCC AGT AG - #C CAA AGC AGC AGT GTT          1224                                                                        Ser Ile Pro Ser Met Ser Ile Ser Ala Ser Se - #r Gln Ser Ser Ser Val                    355          - #       360          - #       365                       - - AAT AGT CTT CCA GAT GCA TCG GAT GAC AAG AG - #T GAG CTA GAC ATG ATG          1272                                                                        Asn Ser Leu Pro Asp Ala Ser Asp Asp Lys Se - #r Glu Leu Asp Met Met                370              - #   375              - #   380                           - - GAG GGA GAC CAT ACA GTG ATG TCT AAC AGT TC - #T GTC ATC CAC TTA AAA          1320                                                                        Glu Gly Asp His Thr Val Met Ser Asn Ser Se - #r Val Ile His Leu Lys            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - CCT GAG GAG GAA AAT TAC CAA GAA GAA GGA GA - #T CCT AGA ACA AGA         GCA     1368                                                                     Pro Glu Glu Glu Asn Tyr Gln Glu Glu Gly As - #p Pro Arg Thr Arg Ala                           405  - #               410  - #               415               - - TCA GCT CCA CAG TCT CCA CCT CAA GTG TCT CG - #T CAC AAA TCA CAT TAT          1416                                                                        Ser Ala Pro Gln Ser Pro Pro Gln Val Ser Ar - #g His Lys Ser His Tyr                        420      - #           425      - #           430                   - - CGT AAT AGA GAA CAC TTT GCA ACT ATA CGA AC - #A GCA TCA CTG GTT ACA          1464                                                                        Arg Asn Arg Glu His Phe Ala Thr Ile Arg Th - #r Ala Ser Leu Val Thr                    435          - #       440          - #       445                       - - AGA CAG ATG CAA GAA CAT GAG CAG GAC TCT GA - #A CTT AGA GAA CAG ATG          1512                                                                        Arg Gln Met Gln Glu His Glu Gln Asp Ser Gl - #u Leu Arg Glu Gln Met                450              - #   455              - #   460                           - - TCT GGT TAT AAG CGG ATG AGG CGA CAG CAT CA - #G AAG CAG CTG ATG ACT          1560                                                                        Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gl - #n Lys Gln Leu Met Thr            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - CTG GAA AAT AAA CTG AAG GCA GAA ATG GAC GA - #A CAT CGG CTC AGA         TTA     1608                                                                     Leu Glu Asn Lys Leu Lys Ala Glu Met Asp Gl - #u His Arg Leu Arg Leu                           485  - #               490  - #               495               - - GAC AAA GAT CTT GAA ACT CAG CGC AAC AAT TT - #C GCT GCA GAA ATG GAG          1656                                                                        Asp Lys Asp Leu Glu Thr Gln Arg Asn Asn Ph - #e Ala Ala Glu Met Glu                        500      - #           505      - #           510                   - - AAA CTT ATT AAG AAA CAC CAA GCT TCT ATG GA - #A AAA GAG GCT AAA GTG          1704                                                                        Lys Leu Ile Lys Lys His Gln Ala Ser Met Gl - #u Lys Glu Ala Lys Val                    515          - #       520          - #       525                       - - ATG GCC AAC GAG GAG AAA AAA TTC CAA CAA CA - #C ATT CAG GCT CAA CAG          1752                                                                        Met Ala Asn Glu Glu Lys Lys Phe Gln Gln Hi - #s Ile Gln Ala Gln Gln                530              - #   535              - #   540                           - - AAG AAA GAA CTG AAT AGC TTT TTG GAG TCT CA - #A AAA AGA GAA TAT AAA          1800                                                                        Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gl - #n Lys Arg Glu Tyr Lys            545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - CTT CGA AAA GAG CAG CTT AAG GAG GAG CTG AA - #T GAA AAC CAG AGC         ACA     1848                                                                     Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu As - #n Glu Asn Gln Ser Thr                           565  - #               570  - #               575               - - CCT AAA AAA GAA AAG CAG GAA TGG CTT TCA AA - #G CAG AAG GAG AAT ATT          1896                                                                        Pro Lys Lys Glu Lys Gln Glu Trp Leu Ser Ly - #s Gln Lys Glu Asn Ile                        580      - #           585      - #           590                   - - CAA CAT TTT CAG GCA GAA GAA GAA GCT AAT CT - #T CTT CGA CGT CAA AGG          1944                                                                        Gln His Phe Gln Ala Glu Glu Glu Ala Asn Le - #u Leu Arg Arg Gln Arg                    595          - #       600          - #       605                       - - CAG TAT CTA GAG CTA GAA TGT CGT CGC TTC AA - #A AGA AGA ATG TTA CTT          1992                                                                        Gln Tyr Leu Glu Leu Glu Cys Arg Arg Phe Ly - #s Arg Arg Met Leu Leu                610              - #   615              - #   620                           - - GGT CGG CAT AAC TTG GAA CAG GAC CTT GTC AG - #G GAG GAG TTA AAC AAA          2040                                                                        Gly Arg His Asn Leu Glu Gln Asp Leu Val Ar - #g Glu Glu Leu Asn Lys            625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - AGG CAG ACT CAG AAG GAC TTA GAA CAT GCA AT - #G TTA CTG CGA CAG         CAT     2088                                                                     Arg Gln Thr Gln Lys Asp Leu Glu His Ala Me - #t Leu Leu Arg Gln His                           645  - #               650  - #               655               - - GAA TCC ATG CAA GAA CTG GAG TTT CGC CAC CT - #C AAC ACT ATT CAG AAG          2136                                                                        Glu Ser Met Gln Glu Leu Glu Phe Arg His Le - #u Asn Thr Ile Gln Lys                        660      - #           665      - #           670                   - - ATG CGC TGT GAG TTG ATC AGA CTG CAA CAT CA - #A ACT GAG CTT ACT AAC          2184                                                                        Met Arg Cys Glu Leu Ile Arg Leu Gln His Gl - #n Thr Glu Leu Thr Asn                    675          - #       680          - #       685                       - - CAG CTG GAA TAC AAT AAG AGA AGG GAA CGG GA - #A CTA AGA CGG AAA CAT          2232                                                                        Gln Leu Glu Tyr Asn Lys Arg Arg Glu Arg Gl - #u Leu Arg Arg Lys His                690              - #   695              - #   700                           - - GTC ATG GAA GTT CGA CAG CAG CCT AAG AGT TT - #G AAG TCT AAA GAA CTC          2280                                                                        Val Met Glu Val Arg Gln Gln Pro Lys Ser Le - #u Lys Ser Lys Glu Leu            705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - CAA ATA AAA AAG CAG TTT CAG GAT ACC TGC AA - #A ATT CAA ACC AGA         CAG     2328                                                                     Gln Ile Lys Lys Gln Phe Gln Asp Thr Cys Ly - #s Ile Gln Thr Arg Gln                           725  - #               730  - #               735               - - TAC AAA GCA TTA AGG AAT CAC CTA CTG GAG AC - #T ACA CCA AAG AGT GAG          2376                                                                        Tyr Lys Ala Leu Arg Asn His Leu Leu Glu Th - #r Thr Pro Lys Ser Glu                        740      - #           745      - #           750                   - - CAC AAA GCT GTT CTG AAA AGA CTC AAG GAG GA - #A CAG ACT CGG AAG TTA          2424                                                                        His Lys Ala Val Leu Lys Arg Leu Lys Glu Gl - #u Gln Thr Arg Lys Leu                    755          - #       760          - #       765                       - - GCC ATC TTG GCT GAG CAG TAT GAT CAT AGC AT - #T AAT GAA ATG CTC TCC          2472                                                                        Ala Ile Leu Ala Glu Gln Tyr Asp His Ser Il - #e Asn Glu Met Leu Ser                770              - #   775              - #   780                           - - ACA CAA GCT CTG CGT TTG GAT GAA GCA CAG GA - #A GCA GAA TGC CAG GTT          2520                                                                        Thr Gln Ala Leu Arg Leu Asp Glu Ala Gln Gl - #u Ala Glu Cys Gln Val            785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - TTG AAG ATG CAG CTA CAG CAG GAA CTG GAG CT - #G TTG AAT GCA TAT         CAG     2568                                                                     Leu Lys Met Gln Leu Gln Gln Glu Leu Glu Le - #u Leu Asn Ala Tyr Gln                           805  - #               810  - #               815               - - AGC AAA ATC AAG ATG CAG GCT GAG GCC CAA CA - #T GAT CGA GAG CTT CGA          2616                                                                        Ser Lys Ile Lys Met Gln Ala Glu Ala Gln Hi - #s Asp Arg Glu Leu Arg                        820      - #           825      - #           830                   - - GAG CTG GAA CAA AGG GTC TCC CTT CGG AGA GC - #A CTC TTA GAA CAG AAG          2664                                                                        Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Al - #a Leu Leu Glu Gln Lys                    835          - #       840          - #       845                       - - ATT GAA GAA GAG ATG TTG GCT TTG CAG AAT GA - #A CGC ACA GAA CGA ATA          2712                                                                        Ile Glu Glu Glu Met Leu Ala Leu Gln Asn Gl - #u Arg Thr Glu Arg Ile                850              - #   855              - #   860                           - - CGT AGC CTG CTC GAG CGC CAG GCC AGA GAA AT - #T GAA GCT TTT GAC TCT          2760                                                                        Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Il - #e Glu Ala Phe Asp Ser            865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - GAA AGC ATG AGA TTA GGT TTT AGT AAC ATG GT - #C CTT TCT AAT CTC         TCC     2808                                                                     Glu Ser Met Arg Leu Gly Phe Ser Asn Met Va - #l Leu Ser Asn Leu Ser                           885  - #               890  - #               895               - - CCT GAG GCA TTC AGC CAC AGC TAC CCA GGA GC - #T TCT AGC TGG TCT CAC          2856                                                                        Pro Glu Ala Phe Ser His Ser Tyr Pro Gly Al - #a Ser Ser Trp Ser His                        900      - #           905      - #           910                   - - AAT CCT ACT GGG GGT TCA GGA CCT CAC TGG GG - #T CAT CCC ATG GGT GGC          2904                                                                        Asn Pro Thr Gly Gly Ser Gly Pro His Trp Gl - #y His Pro Met Gly Gly                    915          - #       920          - #       925                       - - ACA CCA CAA GCT TGG GGT CAT CCG ATG CAA GG - #C GGA CCC CAA CCA TGG          2952                                                                        Thr Pro Gln Ala Trp Gly His Pro Met Gln Gl - #y Gly Pro Gln Pro Trp                930              - #   935              - #   940                           - - GGT CAC CCC TCA GGG CCA ATG CAA GGG GTA CC - #T CGA GGT AGC AGT ATA          3000                                                                        Gly His Pro Ser Gly Pro Met Gln Gly Val Pr - #o Arg Gly Ser Ser Ile            945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - GGA GTC CGC AAT AGC CCC CAG GCT CTG AGG CG - #G ACA GCT TCT GGG         GGA     3048                                                                     Gly Val Arg Asn Ser Pro Gln Ala Leu Arg Ar - #g Thr Ala Ser Gly Gly                           965  - #               970  - #               975               - - CGG ACG GAA CAG GGC ATG AGC AGA AGC ACG AG - #T GTC ACT TCA CAA ATA          3096                                                                        Arg Thr Glu Gln Gly Met Ser Arg Ser Thr Se - #r Val Thr Ser Gln Ile                        980      - #           985      - #           990                   - - TCC AAT GGG TCA CAC ATG TCT TAC ACA TAATAATTG - #A AAGTGGCAAT                3143                                                                        Ser Asn Gly Ser His Met Ser Tyr Thr                                                    995          - #       1000                                             - - TCCGCTGGAG CTGTCTGCCA AAAGAAACTG CCTACAGACA TCAGCACAGC AG -              #CCTCCTCA   3203                                                                  - - CTTGGGTACT ACCGGGTGGA AGCTGTGCAT ATGGTATATT TTATTCGTCT TT -             #GTAAAGCG   3263                                                                  - - TTATGTTTTG TGTTTACTAA TTGGGATGTC ATAGTATTTG GCTGCCGGG  - #                  3312                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1001 amino - #acids                                                (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                            - - Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Ly - #s Asp Pro Glu Ile Ala         1               5 - #                 10 - #                 15               - - Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Le - #u Phe Thr Asp Leu Arg                    20     - #             25     - #             30                   - - Glu Ile Gly His Gly Ser Phe Gly Ala Val Ty - #r Phe Ala Arg Asp Val                35         - #         40         - #         45                       - - Arg Thr Asn Glu Val Val Ala Ile Lys Lys Me - #t Ser Tyr Ser Gly Lys            50             - #     55             - #     60                           - - Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Ly - #s Glu Val Lys Phe Leu        65                 - # 70                 - # 75                 - # 80        - - Gln Arg Ile Lys His Pro Asn Ser Ile Glu Ty - #r Lys Gly Cys Tyr Leu                        85 - #                 90 - #                 95               - - Arg Glu His Thr Ala Trp Leu Val Met Glu Ty - #r Cys Leu Gly Ser Ala                   100      - #           105      - #           110                   - - Ser Asp Leu Leu Glu Val His Lys Lys Pro Le - #u Gln Glu Val Glu Ile               115          - #       120          - #       125                       - - Ala Ala Ile Thr His Gly Ala Leu Gln Gly Le - #u Ala Tyr Leu His Ser           130              - #   135              - #   140                           - - His Thr Met Ile His Arg Asp Ile Lys Ala Gl - #y Asn Ile Leu Leu Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gl - #y Ser Ala Ser Met         Ala                                                                                              165  - #               170  - #               175              - - Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Ty - #r Trp Met Ala Pro Glu                   180      - #           185      - #           190                   - - Val Ile Leu Ala Met Asp Glu Gly Gln Tyr As - #p Gly Lys Val Asp Val               195          - #       200          - #       205                       - - Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Al - #a Glu Arg Lys Pro Pro           210              - #   215              - #   220                           - - Leu Phe Asn Met Asn Ala Met Ser Ala Leu Ty - #r His Ile Ala Gln Asn       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Glu Ser Pro Thr Leu Gln Ser Asn Glu Trp Se - #r Asp Tyr Phe Arg         Asn                                                                                              245  - #               250  - #               255              - - Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gl - #n Asp Arg Pro Thr Ser                   260      - #           265      - #           270                   - - Glu Glu Leu Leu Lys His Met Phe Val Leu Ar - #g Glu Arg Pro Glu Thr               275          - #       280          - #       285                       - - Val Leu Ile Asp Leu Ile Gln Arg Thr Lys As - #p Ala Val Arg Glu Leu           290              - #   295              - #   300                           - - Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Le - #u Leu Phe Gln Glu Ala       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - His Asn Gly Pro Ala Val Glu Ala Gln Glu Gl - #u Glu Glu Glu Gln         Asp                                                                                              325  - #               330  - #               335              - - His Gly Gly Gly Arg Thr Gly Thr Val Asn Se - #r Val Gly Ser Asn Gln                   340      - #           345      - #           350                   - - Ser Ile Pro Ser Met Ser Ile Ser Ala Ser Se - #r Gln Ser Ser Ser Val               355          - #       360          - #       365                       - - Asn Ser Leu Pro Asp Ala Ser Asp Asp Lys Se - #r Glu Leu Asp Met Met           370              - #   375              - #   380                           - - Glu Gly Asp His Thr Val Met Ser Asn Ser Se - #r Val Ile His Leu Lys       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Pro Glu Glu Glu Asn Tyr Gln Glu Glu Gly As - #p Pro Arg Thr Arg         Ala                                                                                              405  - #               410  - #               415              - - Ser Ala Pro Gln Ser Pro Pro Gln Val Ser Ar - #g His Lys Ser His Tyr                   420      - #           425      - #           430                   - - Arg Asn Arg Glu His Phe Ala Thr Ile Arg Th - #r Ala Ser Leu Val Thr               435          - #       440          - #       445                       - - Arg Gln Met Gln Glu His Glu Gln Asp Ser Gl - #u Leu Arg Glu Gln Met           450              - #   455              - #   460                           - - Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gl - #n Lys Gln Leu Met Thr       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Leu Glu Asn Lys Leu Lys Ala Glu Met Asp Gl - #u His Arg Leu Arg         Leu                                                                                              485  - #               490  - #               495              - - Asp Lys Asp Leu Glu Thr Gln Arg Asn Asn Ph - #e Ala Ala Glu Met Glu                   500      - #           505      - #           510                   - - Lys Leu Ile Lys Lys His Gln Ala Ser Met Gl - #u Lys Glu Ala Lys Val               515          - #       520          - #       525                       - - Met Ala Asn Glu Glu Lys Lys Phe Gln Gln Hi - #s Ile Gln Ala Gln Gln           530              - #   535              - #   540                           - - Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gl - #n Lys Arg Glu Tyr Lys       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu As - #n Glu Asn Gln Ser         Thr                                                                                              565  - #               570  - #               575              - - Pro Lys Lys Glu Lys Gln Glu Trp Leu Ser Ly - #s Gln Lys Glu Asn Ile                   580      - #           585      - #           590                   - - Gln His Phe Gln Ala Glu Glu Glu Ala Asn Le - #u Leu Arg Arg Gln Arg               595          - #       600          - #       605                       - - Gln Tyr Leu Glu Leu Glu Cys Arg Arg Phe Ly - #s Arg Arg Met Leu Leu           610              - #   615              - #   620                           - - Gly Arg His Asn Leu Glu Gln Asp Leu Val Ar - #g Glu Glu Leu Asn Lys       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Arg Gln Thr Gln Lys Asp Leu Glu His Ala Me - #t Leu Leu Arg Gln         His                                                                                              645  - #               650  - #               655              - - Glu Ser Met Gln Glu Leu Glu Phe Arg His Le - #u Asn Thr Ile Gln Lys                   660      - #           665      - #           670                   - - Met Arg Cys Glu Leu Ile Arg Leu Gln His Gl - #n Thr Glu Leu Thr Asn               675          - #       680          - #       685                       - - Gln Leu Glu Tyr Asn Lys Arg Arg Glu Arg Gl - #u Leu Arg Arg Lys His           690              - #   695              - #   700                           - - Val Met Glu Val Arg Gln Gln Pro Lys Ser Le - #u Lys Ser Lys Glu Leu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Gln Ile Lys Lys Gln Phe Gln Asp Thr Cys Ly - #s Ile Gln Thr Arg         Gln                                                                                              725  - #               730  - #               735              - - Tyr Lys Ala Leu Arg Asn His Leu Leu Glu Th - #r Thr Pro Lys Ser Glu                   740      - #           745      - #           750                   - - His Lys Ala Val Leu Lys Arg Leu Lys Glu Gl - #u Gln Thr Arg Lys Leu               755          - #       760          - #       765                       - - Ala Ile Leu Ala Glu Gln Tyr Asp His Ser Il - #e Asn Glu Met Leu Ser           770              - #   775              - #   780                           - - Thr Gln Ala Leu Arg Leu Asp Glu Ala Gln Gl - #u Ala Glu Cys Gln Val       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Lys Met Gln Leu Gln Gln Glu Leu Glu Le - #u Leu Asn Ala Tyr         Gln                                                                                              805  - #               810  - #               815              - - Ser Lys Ile Lys Met Gln Ala Glu Ala Gln Hi - #s Asp Arg Glu Leu Arg                   820      - #           825      - #           830                   - - Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Al - #a Leu Leu Glu Gln Lys               835          - #       840          - #       845                       - - Ile Glu Glu Glu Met Leu Ala Leu Gln Asn Gl - #u Arg Thr Glu Arg Ile           850              - #   855              - #   860                           - - Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Il - #e Glu Ala Phe Asp Ser       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Glu Ser Met Arg Leu Gly Phe Ser Asn Met Va - #l Leu Ser Asn Leu         Ser                                                                                              885  - #               890  - #               895              - - Pro Glu Ala Phe Ser His Ser Tyr Pro Gly Al - #a Ser Ser Trp Ser His                   900      - #           905      - #           910                   - - Asn Pro Thr Gly Gly Ser Gly Pro His Trp Gl - #y His Pro Met Gly Gly               915          - #       920          - #       925                       - - Thr Pro Gln Ala Trp Gly His Pro Met Gln Gl - #y Gly Pro Gln Pro Trp           930              - #   935              - #   940                           - - Gly His Pro Ser Gly Pro Met Gln Gly Val Pr - #o Arg Gly Ser Ser Ile       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Gly Val Arg Asn Ser Pro Gln Ala Leu Arg Ar - #g Thr Ala Ser Gly         Gly                                                                                              965  - #               970  - #               975              - - Arg Thr Glu Gln Gly Met Ser Arg Ser Thr Se - #r Val Thr Ser Gln Ile                   980      - #           985      - #           990                   - - Ser Asn Gly Ser His Met Ser Tyr Thr                                               995          - #       1000                                             - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4296 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 193..3171                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                            - - AGGGGAGGCT TCCCGGGCCC GCCCCTCAGG AAGGGCGAAA GCTGAGGAAG AG -              #GTGGCGAG     60                                                                  - - GGGGAAGGTC TCCTTGCCCC TCTCCCCGCT TGTCAGAGCA ACTGGAGTAC CC -             #CAGGCGGA    120                                                                  - - AGCGGAGGCG CTGGGGCACC ATAGTGACCC CTACCAGGCA AGATCCCAAT TT -             #CAGGGCCC    180                                                                  - - CCAGGGGCCA TC ATG CCA GCT GGG GGC CGG GCC GGG - # AGC CTG AAG GAC             228                                                                                     Met Pro Al - #a Gly Gly Arg Ala Gly Ser Leu Lys Asp                              1  - #             5     - #             10                     - - CCT GAT GTA GCT GAG CTC TTC TTC AAA GAT GA - #C CCT GAG AAG CTT TTC           276                                                                        Pro Asp Val Ala Glu Leu Phe Phe Lys Asp As - #p Pro Glu Lys Leu Phe                     15         - #         20         - #         25                       - - TCT GAC CTC CGG GAA ATT GGC CAT GGC AGT TT - #T GGA GCT GTG TAC TTT           324                                                                        Ser Asp Leu Arg Glu Ile Gly His Gly Ser Ph - #e Gly Ala Val Tyr Phe                 30             - #     35             - #     40                           - - GCC CGG GAT GTC CGG AAC AGT GAG GTG GTG GC - #C ATC AAG AAG ATG TCC           372                                                                        Ala Arg Asp Val Arg Asn Ser Glu Val Val Al - #a Ile Lys Lys Met Ser             45                 - # 50                 - # 55                 - # 60        - - TAT AGT GGG AAG CAA TCA AAT GAG AAA TGG CA - #G GAT ATC ATC AAG GAG           420                                                                        Tyr Ser Gly Lys Gln Ser Asn Glu Lys Trp Gl - #n Asp Ile Ile Lys Glu                             65 - #                 70 - #                 75               - - GTG CGG TTC TTA CAG AAG CTA CGG CAT CCT AA - #T ACC ATT CAG TAC CGG           468                                                                        Val Arg Phe Leu Gln Lys Leu Arg His Pro As - #n Thr Ile Gln Tyr Arg                         80     - #             85     - #             90                   - - GGC TGT TAC CTG AGG GAG CAC ACA GCT TGG CT - #G GTG ATG GAG TAT TGC           516                                                                        Gly Cys Tyr Leu Arg Glu His Thr Ala Trp Le - #u Val Met Glu Tyr Cys                     95         - #        100         - #        105                       - - CTG GGT TCA GCT TCT GAT CTT CTC GAA GTG CA - #C AAG AAG CCG CTG CAG           564                                                                        Leu Gly Ser Ala Ser Asp Leu Leu Glu Val Hi - #s Lys Lys Pro Leu Gln                110              - #   115              - #   120                           - - GAG GTA GAG ATT GCA GCT GTG ACC CAT GGT GC - #G CTT CAG GGC CTG GCC           612                                                                        Glu Val Glu Ile Ala Ala Val Thr His Gly Al - #a Leu Gln Gly Leu Ala            125                 1 - #30                 1 - #35                 1 -       #40                                                                               - - TAT CTA CAT TCA CAC AAC ATG ATC CAT AGA GA - #T GTG AAG GCT GGG         AAC      660                                                                     Tyr Leu His Ser His Asn Met Ile His Arg As - #p Val Lys Ala Gly Asn                           145  - #               150  - #               155               - - ATC TTG CTG TCA GAA CCA GGC TTG GTG AAA CT - #G GGG GAC TTT GGC TCC           708                                                                        Ile Leu Leu Ser Glu Pro Gly Leu Val Lys Le - #u Gly Asp Phe Gly Ser                        160      - #           165      - #           170                   - - GCA TCC ATC ATG GCA CCT GCC AAC TCA TTT GT - #G GGC ACT CCA TAC TGG           756                                                                        Ala Ser Ile Met Ala Pro Ala Asn Ser Phe Va - #l Gly Thr Pro Tyr Trp                    175          - #       180          - #       185                       - - ATG GCT CCA GAG GTG ATC CTA GCC ATG GAT GA - #G GGA CAA TAT GAT GGC           804                                                                        Met Ala Pro Glu Val Ile Leu Ala Met Asp Gl - #u Gly Gln Tyr Asp Gly                190              - #   195              - #   200                           - - AAA GTG GAT GTC TGG TCC TTG GGG ATA ACC TG - #T ATT GAG CTA GCG GAG           852                                                                        Lys Val Asp Val Trp Ser Leu Gly Ile Thr Cy - #s Ile Glu Leu Ala Glu            205                 2 - #10                 2 - #15                 2 -       #20                                                                               - - CGG AAG CCA CCA CTG TTT AAC ATG AAT GCA AT - #G AGT GCC TTA TAC         CAC      900                                                                     Arg Lys Pro Pro Leu Phe Asn Met Asn Ala Me - #t Ser Ala Leu Tyr His                           225  - #               230  - #               235               - - ATT GCA CAG AAT GAA TCC CCT GCT CTC CAG TC - #A GGA CAC TGG TCT GAG           948                                                                        Ile Ala Gln Asn Glu Ser Pro Ala Leu Gln Se - #r Gly His Trp Ser Glu                        240      - #           245      - #           250                   - - TAC TTC CGG AAT TTT GTT GAC TCC TGT CTT CA - #G AAA ATC CCT CAA GAC           996                                                                        Tyr Phe Arg Asn Phe Val Asp Ser Cys Leu Gl - #n Lys Ile Pro Gln Asp                    255          - #       260          - #       265                       - - AGA CCA ACC TCA GAG GTT CTT TTG AAG CAC CG - #C TTT GTG CTC CGG GAG          1044                                                                        Arg Pro Thr Ser Glu Val Leu Leu Lys His Ar - #g Phe Val Leu Arg Glu                270              - #   275              - #   280                           - - CGG CCA CCC ACA GTC ATC ATG GAC CTA ATT CA - #G AGG ACC AAG GAT GCT          1092                                                                        Arg Pro Pro Thr Val Ile Met Asp Leu Ile Gl - #n Arg Thr Lys Asp Ala            285                 2 - #90                 2 - #95                 3 -       #00                                                                               - - GTA CGG GAA CTA GAT AAC CTG CAG TAC CGA AA - #G ATG AAG AAG ATA         CTA     1140                                                                     Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg Ly - #s Met Lys Lys Ile Leu                           305  - #               310  - #               315               - - TTC CAA GAG GCA CCC AAT GGC CCT GGT GCT GA - #G GCC CCA GAG GAA GAG          1188                                                                        Phe Gln Glu Ala Pro Asn Gly Pro Gly Ala Gl - #u Ala Pro Glu Glu Glu                        320      - #           325      - #           330                   - - GAG GAA GCA GAA CCT TAC ATG CAC CGA GCA GG - #G ACA CTG ACC AGT CTA          1236                                                                        Glu Glu Ala Glu Pro Tyr Met His Arg Ala Gl - #y Thr Leu Thr Ser Leu                    335          - #       340          - #       345                       - - GAG AGT AGC CAT TCA GTG CCC AGC ATG TCC AT - #C AGC GCC TCC AGC CAA          1284                                                                        Glu Ser Ser His Ser Val Pro Ser Met Ser Il - #e Ser Ala Ser Ser Gln                350              - #   355              - #   360                           - - AGC AGC TCA GTC AAC AGC CTA GCA GAT GCC TC - #A GAT AAT GAA GAA GAG          1332                                                                        Ser Ser Ser Val Asn Ser Leu Ala Asp Ala Se - #r Asp Asn Glu Glu Glu            365                 3 - #70                 3 - #75                 3 -       #80                                                                               - - GAG GAG GAG GAA GAG GAA GAA GAA GAG GAG GA - #G GAA GAA GAA GGC         CCT     1380                                                                     Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gl - #u Glu Glu Glu Gly Pro                           385  - #               390  - #               395               - - GAA TCC CGA GAG ATG GCC ATG ATG CAG GAG GG - #G GAG CAT ACA GTC ACT          1428                                                                        Glu Ser Arg Glu Met Ala Met Met Gln Glu Gl - #y Glu His Thr Val Thr                        400      - #           405      - #           410                   - - TCC CAC AGC TCC ATC ATC CAC CGG CTG CCG GG - #C TCA GAC AAC CTA TAT          1476                                                                        Ser His Ser Ser Ile Ile His Arg Leu Pro Gl - #y Ser Asp Asn Leu Tyr                    415          - #       420          - #       425                       - - GAT GAT CCC TAC CAG CCA GAG ATG ACC CCA GG - #T CCA CTC CAA CCA CCT          1524                                                                        Asp Asp Pro Tyr Gln Pro Glu Met Thr Pro Gl - #y Pro Leu Gln Pro Pro                430              - #   435              - #   440                           - - GCA GCC CCT CCC ACC TCC ACC TCC TCC TCT TC - #T GCT CGC CGC AGA GCT          1572                                                                        Ala Ala Pro Pro Thr Ser Thr Ser Ser Ser Se - #r Ala Arg Arg Arg Ala            445                 4 - #50                 4 - #55                 4 -       #60                                                                               - - TAT TGC CGC AAC CGA GAC CAC TTT GCC ACC AT - #C CGT ACT GCC TCC         CTG     1620                                                                     Tyr Cys Arg Asn Arg Asp His Phe Ala Thr Il - #e Arg Thr Ala Ser Leu                           465  - #               470  - #               475               - - GTC AGC CGT CAG ATC CAG GAG CAT GAG CAG GA - #C TCG GCC CTG CGG GAG          1668                                                                        Val Ser Arg Gln Ile Gln Glu His Glu Gln As - #p Ser Ala Leu Arg Glu                        480      - #           485      - #           490                   - - CAA CTA AGT GGC TAC AAG CGG ATG CGG CGT CA - #G CAC CAG AAG CAA CTG          1716                                                                        Gln Leu Ser Gly Tyr Lys Arg Met Arg Arg Gl - #n His Gln Lys Gln Leu                    495          - #       500          - #       505                       - - CTG GCC CTG GAG TCC CGT CTG AGG GGT GAA CG - #T GAG GAG CAC AGT GGG          1764                                                                        Leu Ala Leu Glu Ser Arg Leu Arg Gly Glu Ar - #g Glu Glu His Ser Gly                510              - #   515              - #   520                           - - CGG TTG CAG CGT GAA CTC GAG GCA CAG CGG GC - #T GGC TTT GGG ACT GAG          1812                                                                        Arg Leu Gln Arg Glu Leu Glu Ala Gln Arg Al - #a Gly Phe Gly Thr Glu            525                 5 - #30                 5 - #35                 5 -       #40                                                                               - - GCT GAG AAG CTG GCC CGG AGG CAC CAG GCC AT - #T GGT GAG AAG GAA         GCA     1860                                                                     Ala Glu Lys Leu Ala Arg Arg His Gln Ala Il - #e Gly Glu Lys Glu Ala                           545  - #               550  - #               555               - - CGA GCT GCT CAG GCT GAG GAG CGG AAG TTC CA - #G CAG CAC ATC TTG GGG          1908                                                                        Arg Ala Ala Gln Ala Glu Glu Arg Lys Phe Gl - #n Gln His Ile Leu Gly                        560      - #           565      - #           570                   - - CAG CAG AAG AAG GAA CTG GCT GCC CTG CTG GA - #G GCA CAG AAG CGA ACC          1956                                                                        Gln Gln Lys Lys Glu Leu Ala Ala Leu Leu Gl - #u Ala Gln Lys Arg Thr                    575          - #       580          - #       585                       - - TAT AAG CTT CGG AAG GAG CAG TTG AAA GAG GA - #G CTC CAG GAG AAC CCT          2004                                                                        Tyr Lys Leu Arg Lys Glu Gln Leu Lys Glu Gl - #u Leu Gln Glu Asn Pro                590              - #   595              - #   600                           - - AGC ACA CCC AAA CGA GAG AAG GCT GAG TGG CT - #G TTG AGG CAG AAA GAG          2052                                                                        Ser Thr Pro Lys Arg Glu Lys Ala Glu Trp Le - #u Leu Arg Gln Lys Glu            605                 6 - #10                 6 - #15                 6 -       #20                                                                               - - CAG TTG CAA CAG TGC CAG GCA GAG GAG GAG GC - #A GGG CTA CTG CGG         AGG     2100                                                                     Gln Leu Gln Gln Cys Gln Ala Glu Glu Glu Al - #a Gly Leu Leu Arg Arg                           625  - #               630  - #               635               - - CAA CGC CAG TAC TTT GAG CTT CAG TGT CGC CA - #A TAC AAG CGC AAG ATG          2148                                                                        Gln Arg Gln Tyr Phe Glu Leu Gln Cys Arg Gl - #n Tyr Lys Arg Lys Met                        640      - #           645      - #           650                   - - CTA CTG GCT CGG CAC AGC CTA GAC CAG GAC CT - #G CTT CGA GAG GAC TTG          2196                                                                        Leu Leu Ala Arg His Ser Leu Asp Gln Asp Le - #u Leu Arg Glu Asp Leu                    655          - #       660          - #       665                       - - AAT AAG AAA CAG ACA CAG AAG GAC TTG GAG TG - #T GCT CTG CTG TTA CGG          2244                                                                        Asn Lys Lys Gln Thr Gln Lys Asp Leu Glu Cy - #s Ala Leu Leu Leu Arg                670              - #   675              - #   680                           - - CAG CAT GAG GCT ACC CGA GAG CTG GAG CTA CG - #A CAG CTC CAG GCT GTC          2292                                                                        Gln His Glu Ala Thr Arg Glu Leu Glu Leu Ar - #g Gln Leu Gln Ala Val            685                 6 - #90                 6 - #95                 7 -       #00                                                                               - - CAG CGC ACA CGT GCT GAA CTC ACC CGC CTT CA - #G CAC CAG ACA GAG         CTA     2340                                                                     Gln Arg Thr Arg Ala Glu Leu Thr Arg Leu Gl - #n His Gln Thr Glu Leu                           705  - #               710  - #               715               - - GGC AAC CAG TTG GAG TAC AAC AAG CGA CGG GA - #G CAA GAG TTG CGG CAG          2388                                                                        Gly Asn Gln Leu Glu Tyr Asn Lys Arg Arg Gl - #u Gln Glu Leu Arg Gln                        720      - #           725      - #           730                   - - AAG CAC GCG GCC CAG GTT CGC CAG CAG CCC AA - #G AGC CTC AAA GTA CGT          2436                                                                        Lys His Ala Ala Gln Val Arg Gln Gln Pro Ly - #s Ser Leu Lys Val Arg                    735          - #       740          - #       745                       - - GCA GGC CAG CTA CCC ATG GGC CTC CCT GCT AC - #C GGG GCT CTG GGA CCA          2484                                                                        Ala Gly Gln Leu Pro Met Gly Leu Pro Ala Th - #r Gly Ala Leu Gly Pro                750              - #   755              - #   760                           - - CTC AGC ACA GGC ACC CTT AGT GAA GAG CAG CC - #C TGC TCA TCT GGC CAG          2532                                                                        Leu Ser Thr Gly Thr Leu Ser Glu Glu Gln Pr - #o Cys Ser Ser Gly Gln            765                 7 - #70                 7 - #75                 7 -       #80                                                                               - - GAG GCA ATC CTG GGC CAA AGG ATG CTG GGA GA - #G GAG GAG GAA GCA         GTG     2580                                                                     Glu Ala Ile Leu Gly Gln Arg Met Leu Gly Gl - #u Glu Glu Glu Ala Val                           785  - #               790  - #               795               - - CCA GAG AGA ATG ATT CTG GGA AAG GAA GGG AC - #T ACT TTG GAG CCA GAG          2628                                                                        Pro Glu Arg Met Ile Leu Gly Lys Glu Gly Th - #r Thr Leu Glu Pro Glu                        800      - #           805      - #           810                   - - GAG CAG AGG ATT CTG GGG GAA GAA ATG GGA AC - #C TTT AGT TCC AGC CCA          2676                                                                        Glu Gln Arg Ile Leu Gly Glu Glu Met Gly Th - #r Phe Ser Ser Ser Pro                    815          - #       820          - #       825                       - - CAA AAA CAT AGG AGT CTG GTT AAT GAG GAA GA - #T TGG GAT ATA TCT AAA          2724                                                                        Gln Lys His Arg Ser Leu Val Asn Glu Glu As - #p Trp Asp Ile Ser Lys                830              - #   835              - #   840                           - - GAA ATG AAG GAG AGT AGA GTC CCA TCC CTG GC - #A TCC CAG GAG AGA AAT          2772                                                                        Glu Met Lys Glu Ser Arg Val Pro Ser Leu Al - #a Ser Gln Glu Arg Asn            845                 8 - #50                 8 - #55                 8 -       #60                                                                               - - ATT ATT GGC CAG GAA GAG GCT GGG GCA TGG AA - #T CTG TGG GAG AAG         GAG     2820                                                                     Ile Ile Gly Gln Glu Glu Ala Gly Ala Trp As - #n Leu Trp Glu Lys Glu                           865  - #               870  - #               875               - - CAT GGA AAC CTT GTG GAT ATG GAG TTC AAG CT - #T GGC TGG GTC CAG GGT          2868                                                                        His Gly Asn Leu Val Asp Met Glu Phe Lys Le - #u Gly Trp Val Gln Gly                        880      - #           885      - #           890                   - - CCA GTT CTG ACT CCA GTG CCT GAG GAG GAA GA - #G GAG GAG GAA GAG GAG          2916                                                                        Pro Val Leu Thr Pro Val Pro Glu Glu Glu Gl - #u Glu Glu Glu Glu Glu                    895          - #       900          - #       905                       - - GGA GGG GCT CCA ATT GGA ACC CCC AGG GAC CC - #T GGA GAT GGC TGT CCT          2964                                                                        Gly Gly Ala Pro Ile Gly Thr Pro Arg Asp Pr - #o Gly Asp Gly Cys Pro                910              - #   915              - #   920                           - - TCC CCA GAT ATC CCC CCA GAG CCA CCT CCA TC - #A CAT CTG AGA CAG TAC          3012                                                                        Ser Pro Asp Ile Pro Pro Glu Pro Pro Pro Se - #r His Leu Arg Gln Tyr            925                 9 - #30                 9 - #35                 9 -       #40                                                                               - - CCT GCT AGC CAG CTT CCT GGA TTC TTG TCT CA - #T GGC CTC CTG ACT         GGC     3060                                                                     Pro Ala Ser Gln Leu Pro Gly Phe Leu Ser Hi - #s Gly Leu Leu Thr Gly                           945  - #               950  - #               955               - - CTC TCC TTT GCA GTG GGG TCC TCC TCT GGC CT - #C TTG CCC CTA CTA CTT          3108                                                                        Leu Ser Phe Ala Val Gly Ser Ser Ser Gly Le - #u Leu Pro Leu Leu Leu                        960      - #           965      - #           970                   - - CTG CTG CTA CTC CCA TTG CTG GCA CCC AGG TG - #G AGG TGG CTT GCA GGC          3156                                                                        Leu Leu Leu Leu Pro Leu Leu Ala Pro Arg Tr - #p Arg Trp Leu Ala Gly                    975          - #       980          - #       985                       - - AGC ACT GCT GGC CCT T GAGGTAGGAC TAGTGGGCCT GGGG - #GCTTCA                   3202                                                                        Ser Thr Ala Gly Pro                                                                990                                                                         - - TACCTGTTCC TTTGTACAGC TCTACACCTG CCACCCAGTC TGTTCTTACT CC -              #TGGCTCAG   3262                                                                  - - GGCACTGCAC TGGGGGCTGT CCTTAGCCTG AGCTGGCGCA GAGGCCTTAT GG -             #GTGTGCCT   3322                                                                  - - CTGGGCCTTG GGGCTGCCTG GCTCCTAGCT TGGCCCAGCC TGGCTTTACC TC -             #TGGCAGCT   3382                                                                  - - ATGGCGGCTG GGGGCAAATG GGTACGGCAG CAAGGCCCCC AGATGCGTCG GG -             #GCATCTCT   3442                                                                  - - CGACTCTGGT TGCGGGTTCT GCTACGCCTG TCACCCATGG TCTTTCGGGC CC -             #TACAGGGC   3502                                                                  - - TGTGCGGCTG TGGGAGACCG GGGGCTGTTT GCCCTGTACC CTAAGACCAA TA -             #AGAATGGT   3562                                                                  - - TTCCGAAGTC GACTGCCTGT CCCTTGGCCC CGTCAGGGAA ATCCTCGCAC TA -             #CACAGCAC   3622                                                                  - - CCACTAGCTC TGTTAGCAAG AGTTTGGGCT CTGTGCAAGG GCTGGAACTG GC -             #GCCTAGCA   3682                                                                  - - CGGGCTAGCC ATAGATTAGC TTCTTGTTTG CCCCCCTGGG CTGTTCATAT AC -             #TAGCTAGC   3742                                                                  - - TGGGGCCTGC TTAAGGGTGA AAGGCCCAGT CGGATCCCTC GGCTGCTACC GC -             #GAAGCCAA   3802                                                                  - - CGCCGTCTTG GGCTCTCAGC TTCCCGACAG CTACCACCAG GGACTGTAGC TG -             #GGCGGAGA   3862                                                                  - - TCTCAGACCC GCAGGGCCCT GCCTCCCTGG AGGTAACCAG TTCTAACCCT CC -             #ACCCAAAT   3922                                                                  - - TTAGGGCATT GAGCACTTTA TCTCCCATGA CTCAGTAAAG TCTCTCCAGT CC -             #CTTGGCCT   3982                                                                  - - CTCCTCCCCT TCTGACCTTT CTTCCTCAGT ATGTTTCCCC AGGTCCAATC CC -             #AGCCCCAG   4042                                                                  - - ATGTAGATTT CTAGACAGGC AGCCTCCTCT ACTGTGGAGT CCAGAATGAC AC -             #TCTTGTGT   4102                                                                  - - TTTCCCCAGT CCCCTAAGTT ATTGCTGTCC CCTGCTGTGT GTGTGCTCAT CC -             #TCACCCTC   4162                                                                  - - ATCGGCTCAG GCCTGGGGCC AGGGGTGGCA GGGAGGGAAG TCATGGGGGT TT -             #TCCCTCTT   4222                                                                  - - TGATTTTGTT TTTCTGTCTC CCTTCCAACC TGTCCCCTTC CCCTCCACCA AA -             #AGAGAAAA   4282                                                                  - - AAAAAAAAAA AAAA              - #                  - #                       - #   4296                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 993 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                            - - Met Pro Ala Gly Gly Arg Ala Gly Ser Leu Ly - #s Asp Pro Asp Val Ala         1               5 - #                 10 - #                 15               - - Glu Leu Phe Phe Lys Asp Asp Pro Glu Lys Le - #u Phe Ser Asp Leu Arg                    20     - #             25     - #             30                   - - Glu Ile Gly His Gly Ser Phe Gly Ala Val Ty - #r Phe Ala Arg Asp Val                35         - #         40         - #         45                       - - Arg Asn Ser Glu Val Val Ala Ile Lys Lys Me - #t Ser Tyr Ser Gly Lys            50             - #     55             - #     60                           - - Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Ly - #s Glu Val Arg Phe Leu        65                 - # 70                 - # 75                 - # 80        - - Gln Lys Leu Arg His Pro Asn Thr Ile Gln Ty - #r Arg Gly Cys Tyr Leu                        85 - #                 90 - #                 95               - - Arg Glu His Thr Ala Trp Leu Val Met Glu Ty - #r Cys Leu Gly Ser Ala                   100      - #           105      - #           110                   - - Ser Asp Leu Leu Glu Val His Lys Lys Pro Le - #u Gln Glu Val Glu Ile               115          - #       120          - #       125                       - - Ala Ala Val Thr His Gly Ala Leu Gln Gly Le - #u Ala Tyr Leu His Ser           130              - #   135              - #   140                           - - His Asn Met Ile His Arg Asp Val Lys Ala Gl - #y Asn Ile Leu Leu Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Glu Pro Gly Leu Val Lys Leu Gly Asp Phe Gl - #y Ser Ala Ser Ile         Met                                                                                              165  - #               170  - #               175              - - Ala Pro Ala Asn Ser Phe Val Gly Thr Pro Ty - #r Trp Met Ala Pro Glu                   180      - #           185      - #           190                   - - Val Ile Leu Ala Met Asp Glu Gly Gln Tyr As - #p Gly Lys Val Asp Val               195          - #       200          - #       205                       - - Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Al - #a Glu Arg Lys Pro Pro           210              - #   215              - #   220                           - - Leu Phe Asn Met Asn Ala Met Ser Ala Leu Ty - #r His Ile Ala Gln Asn       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Glu Ser Pro Ala Leu Gln Ser Gly His Trp Se - #r Glu Tyr Phe Arg         Asn                                                                                              245  - #               250  - #               255              - - Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gl - #n Asp Arg Pro Thr Ser                   260      - #           265      - #           270                   - - Glu Val Leu Leu Lys His Arg Phe Val Leu Ar - #g Glu Arg Pro Pro Thr               275          - #       280          - #       285                       - - Val Ile Met Asp Leu Ile Gln Arg Thr Lys As - #p Ala Val Arg Glu Leu           290              - #   295              - #   300                           - - Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Il - #e Leu Phe Gln Glu Ala       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Pro Asn Gly Pro Gly Ala Glu Ala Pro Glu Gl - #u Glu Glu Glu Ala         Glu                                                                                              325  - #               330  - #               335              - - Pro Tyr Met His Arg Ala Gly Thr Leu Thr Se - #r Leu Glu Ser Ser His                   340      - #           345      - #           350                   - - Ser Val Pro Ser Met Ser Ile Ser Ala Ser Se - #r Gln Ser Ser Ser Val               355          - #       360          - #       365                       - - Asn Ser Leu Ala Asp Ala Ser Asp Asn Glu Gl - #u Glu Glu Glu Glu Glu           370              - #   375              - #   380                           - - Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gl - #y Pro Glu Ser Arg Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Met Ala Met Met Gln Glu Gly Glu His Thr Va - #l Thr Ser His Ser         Ser                                                                                              405  - #               410  - #               415              - - Ile Ile His Arg Leu Pro Gly Ser Asp Asn Le - #u Tyr Asp Asp Pro Tyr                   420      - #           425      - #           430                   - - Gln Pro Glu Met Thr Pro Gly Pro Leu Gln Pr - #o Pro Ala Ala Pro Pro               435          - #       440          - #       445                       - - Thr Ser Thr Ser Ser Ser Ser Ala Arg Arg Ar - #g Ala Tyr Cys Arg Asn           450              - #   455              - #   460                           - - Arg Asp His Phe Ala Thr Ile Arg Thr Ala Se - #r Leu Val Ser Arg Gln       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Ile Gln Glu His Glu Gln Asp Ser Ala Leu Ar - #g Glu Gln Leu Ser         Gly                                                                                              485  - #               490  - #               495              - - Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gl - #n Leu Leu Ala Leu Glu                   500      - #           505      - #           510                   - - Ser Arg Leu Arg Gly Glu Arg Glu Glu His Se - #r Gly Arg Leu Gln Arg               515          - #       520          - #       525                       - - Glu Leu Glu Ala Gln Arg Ala Gly Phe Gly Th - #r Glu Ala Glu Lys Leu           530              - #   535              - #   540                           - - Ala Arg Arg His Gln Ala Ile Gly Glu Lys Gl - #u Ala Arg Ala Ala Gln       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Ala Glu Glu Arg Lys Phe Gln Gln His Ile Le - #u Gly Gln Gln Lys         Lys                                                                                              565  - #               570  - #               575              - - Glu Leu Ala Ala Leu Leu Glu Ala Gln Lys Ar - #g Thr Tyr Lys Leu Arg                   580      - #           585      - #           590                   - - Lys Glu Gln Leu Lys Glu Glu Leu Gln Glu As - #n Pro Ser Thr Pro Lys               595          - #       600          - #       605                       - - Arg Glu Lys Ala Glu Trp Leu Leu Arg Gln Ly - #s Glu Gln Leu Gln Gln           610              - #   615              - #   620                           - - Cys Gln Ala Glu Glu Glu Ala Gly Leu Leu Ar - #g Arg Gln Arg Gln Tyr       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Phe Glu Leu Gln Cys Arg Gln Tyr Lys Arg Ly - #s Met Leu Leu Ala         Arg                                                                                              645  - #               650  - #               655              - - His Ser Leu Asp Gln Asp Leu Leu Arg Glu As - #p Leu Asn Lys Lys Gln                   660      - #           665      - #           670                   - - Thr Gln Lys Asp Leu Glu Cys Ala Leu Leu Le - #u Arg Gln His Glu Ala               675          - #       680          - #       685                       - - Thr Arg Glu Leu Glu Leu Arg Gln Leu Gln Al - #a Val Gln Arg Thr Arg           690              - #   695              - #   700                           - - Ala Glu Leu Thr Arg Leu Gln His Gln Thr Gl - #u Leu Gly Asn Gln Leu       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Glu Tyr Asn Lys Arg Arg Glu Gln Glu Leu Ar - #g Gln Lys His Ala         Ala                                                                                              725  - #               730  - #               735              - - Gln Val Arg Gln Gln Pro Lys Ser Leu Lys Va - #l Arg Ala Gly Gln Leu                   740      - #           745      - #           750                   - - Pro Met Gly Leu Pro Ala Thr Gly Ala Leu Gl - #y Pro Leu Ser Thr Gly               755          - #       760          - #       765                       - - Thr Leu Ser Glu Glu Gln Pro Cys Ser Ser Gl - #y Gln Glu Ala Ile Leu           770              - #   775              - #   780                           - - Gly Gln Arg Met Leu Gly Glu Glu Glu Glu Al - #a Val Pro Glu Arg Met       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Ile Leu Gly Lys Glu Gly Thr Thr Leu Glu Pr - #o Glu Glu Gln Arg         Ile                                                                                              805  - #               810  - #               815              - - Leu Gly Glu Glu Met Gly Thr Phe Ser Ser Se - #r Pro Gln Lys His Arg                   820      - #           825      - #           830                   - - Ser Leu Val Asn Glu Glu Asp Trp Asp Ile Se - #r Lys Glu Met Lys Glu               835          - #       840          - #       845                       - - Ser Arg Val Pro Ser Leu Ala Ser Gln Glu Ar - #g Asn Ile Ile Gly Gln           850              - #   855              - #   860                           - - Glu Glu Ala Gly Ala Trp Asn Leu Trp Glu Ly - #s Glu His Gly Asn Leu       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Val Asp Met Glu Phe Lys Leu Gly Trp Val Gl - #n Gly Pro Val Leu         Thr                                                                                              885  - #               890  - #               895              - - Pro Val Pro Glu Glu Glu Glu Glu Glu Glu Gl - #u Glu Gly Gly Ala Pro                   900      - #           905      - #           910                   - - Ile Gly Thr Pro Arg Asp Pro Gly Asp Gly Cy - #s Pro Ser Pro Asp Ile               915          - #       920          - #       925                       - - Pro Pro Glu Pro Pro Pro Ser His Leu Arg Gl - #n Tyr Pro Ala Ser Gln           930              - #   935              - #   940                           - - Leu Pro Gly Phe Leu Ser His Gly Leu Leu Th - #r Gly Leu Ser Phe Ala       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Val Gly Ser Ser Ser Gly Leu Leu Pro Leu Le - #u Leu Leu Leu Leu         Leu                                                                                              965  - #               970  - #               975              - - Pro Leu Leu Ala Pro Arg Trp Arg Trp Leu Al - #a Gly Ser Thr Ala Gly                   980      - #           985      - #           990                   - - Pro                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 414 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                            - - ACGANTCACC AGTTGGAAGT TACTCCAAAG AATGAGCACA AAACAATCTT AA -              #AGACACTG     60                                                                  - - AAAGATGAGC AGACAAGAAA ACTTGCCATT TNGGCAGAGC AGTATGAACA GA -             #GTATAAAT    120                                                                  - - GAAATGATGG CCTCTCANGC GTTACGGCTA GATGAGGCTC AAGAAGCAGA AT -             #GCCAGGCC    180                                                                  - - TTGAGGCTAC AGCTCCAGCA GGAAATGGAG CTGCTCAACG CCTACCAGAG CA -             #AAATCAAG    240                                                                  - - ATGCAAACAG AGGCACAACA TGAACGTGAG CTCCAGAAGC TAGAGCAGAG AG -             #TGTCTCTG    300                                                                  - - CGCAGAGCAC ACCTTGAGCA GAAGATTGAA GAGGAGCTGG CTGCCCTTCA GA -             #AGGAACGC    360                                                                  - - AGCGAGAGAA TAAAGAACCT ATTGGAAAGG CAAGAGCGAG AGATTGGAAA CT - #TT               414                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 314 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                            - - GAACAAAGTC ATGCCTTAAT AGTTCTGCTG ATGTTGGCCT TTCCTGAGGT AT -              #TTTCTGCA     60                                                                  - - AGCAGTAATC AACAAATCTC CTAAAGGAGT CTGTCCATTC ATTAGACTGT AA -             #CGTTGGGG    120                                                                  - - AGTCATTCTG GGCAATGTGA TATAAGGCAC TCATTGCATT CATGTTGAAA AG -             #GGGCGGCT    180                                                                  - - TCCGTTCCGC CAATTCAATA CAAGTGATGC CAAGTGACCA AATATCAACT TT -             #CCCATCAT    240                                                                  - - ACTGTCCTTC ATCCATAGCT AAGATCACCT CTGGAGCCAT CCAGTAAGGT GT -             #GCCCACGA    300                                                                  - - AGGAGTTGGC CAGG              - #                  - #                       - #    314                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 370 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                            - - ACCAAATTCC CAAATCCCAT TCTGAGGCTC TCCATGTCAA AAGTTTCAAT CT -              #CTCGCTCT     60                                                                  - - TGCCTTTCCA ATAGGTTCTT TATTCTCTCG CTGCGTTCCT TCTGAAGGGC AG -             #CCAGCTCC    120                                                                  - - TCTTCAATCT TCTGCTCAAG GTGTGGTCTG CGCAGAGACA CTCTCTGCTC TA -             #GCTTCTGG    180                                                                  - - AGCTCACGTT CATGTTGTGC CTCTGTTNGN ATCTTGATTT GGNTCTGGTA GG -             #CGTTGAGC    240                                                                  - - AGCTCCATTT CCTGCTGGAG CTGTAGCCTC AAGGCCTGGC ATTCTGCTTC TT -             #GAGCCTCA    300                                                                  - - TCTAGCCGTA ACGCTTGAGA GGCCATCATT TCATTTATAC TCTGTTCATA CT -             #GCTCTGCC    360                                                                  - - AAAATGGCAA                - #                  - #                       - #       370                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 190 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                            - - CAACAGCAGA AAAACTTAAA GGCCATGGAA ATGCAAATTA AAAAACAGTT TC -              #AGGACACT     60                                                                  - - TGCAAAGTAC AGACCAAACA GTATAAAGCA CTCAAGAATC ACCAGTTGGA AG -             #TTACTCCA    120                                                                  - - AAGAATGAGC ACAAAACAAT CTTAAAGACA CTGAAAGATG AGCAGACAAG AA -             #AACTTGCC    180                                                                  - - ATTTTGGCAG                - #                  - #                       - #       190                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 65 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                            - - GAGCAGTATG AACAGAGTAT AAATGAAATG ATGGCCTCTC AAGCGTTACG GC -              #TAGATGAG     60                                                                  - - GCTCA                 - #                  - #                  -       #            65                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 219 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                           - - ACGAGTCCCC CCGAGAGCTA GAGTACAGGC AGCTGCACAC GTTACAGAAG CT -             #ACGCATGG     60                                                                  - - ATCTGATCCG TTTACAGCAC CAGACGGAAC TGGAAAACCA GCTGGAGTAC AA -             #TAAGAGGC    120                                                                  - - GAGAAAGAGA ACTGCACAGA AAGCATGTCA TGGAACTTCG GCAACAGCCA AA -             #AAACTTAA    180                                                                  - - AGGCCATGGA ANTGCAATTT AAAAAACAGT TCCAGGAAA      - #                       - #   219                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 85 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                           - - GTGCATATGG TATATTTNAT TCATTTTTGT AAAGCGTTCT GTTTTGTGTT TA -              #CTAATTGG     60                                                                  - - GATGTCATAG TACTTGGCTG CCGGG          - #                  - #                    85                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 46 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                           - - CTCACTTGGG TACTACAGTG TGGAAGCTGA GTGCATATGG TATATT   - #                      46                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 116 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                           - - GATATTTGGT CATTGGGTAT CACGTGTATA GAGCTGGCCG AACGTCGTCC AC -              #CATTGTTC     60                                                                  - - AGTATGAATG CAATGTCTGC CCTCTACCAT ATTGCTCAAA ATGATCCTCC AA - #CTCT             116                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 118 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                           - - CTGAAAGGCC TGGATTATCT GCACTCAGAG CGCAAGATCC ACCGAGATAT CA -              #AAGCTGCC     60                                                                  - - AACGTGCTGC TCTCGGAGCA GGGTGATGTG AAGATGGCAG ACTTCGGTGT GG -             #CTGGCA      118                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 110 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                           - - GACCCAGAGG AACTCTTCAC CAAGCTTGAC CGCATTGGCA AAGGCTCATT TG -             #GGGAGGTG     60                                                                  - - TACAAGGGGA TCGACAACCA CACCAAGGAA GTGGTGGCCA TCAAGATCAT  - #                  110                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 134 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                           - - TCAGGATTCT GGAGCTCTGG AGTTCCATTA GTGGCTATCA GATACAATGC CC -              #TGAGTGGA     60                                                                  - - TTTTCATTAA GGTAAGGGGG TTCACCTTCC ACCATTTCAA TTGCCATAAT TC -             #CAAGAGAC    120                                                                  - - CAGATATCAA CTTT              - #                  - #                       - #    134                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 278 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                           - - Met Ala Pro Ala Val Leu Gln Lys Pro Gly Va - #l Ile Lys Asp Pro Ser       1               5   - #                10  - #                15                - - Ile Ala Ala Leu Phe Ser Asn Lys Asp Pro Gl - #u Gln Asp Leu Arg Glu                   20      - #            25      - #            30                    - - Ile Gly His Gly Ser Phe Gly Ala Val Tyr Ph - #e Ala Tyr Asp Lys Lys               35          - #        40          - #        45                        - - Asn Glu Gln Thr Val Ala Ile Lys Lys Met As - #n Phe Ser Gly Lys Gln           50              - #    55              - #    60                            - - Ala Val Glu Lys Trp Asn Asp Ile Leu Lys Gl - #u Val Ser Phe Leu Asn       65                  - #70                  - #75                  - #80         - - Thr Val Val His Pro His Ile Val Asp Tyr Ly - #s Ala Cys Phe Leu Lys                       85  - #                90  - #                95                - - Asp Thr Thr Cys Trp Leu Val Met Glu Tyr Cy - #s Ile Gly Ser Ala Ala                   100      - #           105      - #           110                   - - Asp Ile Val Asp Val Leu Arg Lys Gly Met Ar - #g Glu Val Glu Ile Ala               115          - #       120          - #       125                       - - Ala Ile Cys Ser Gln Thr Leu Asp Ala Leu Ar - #g Tyr Leu His Ser Leu           130              - #   135              - #   140                           - - Lys Arg Ile His Arg Asp Ile Lys Ala Gly As - #n Ile Leu Leu Ser Asp       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - His Ala Ile Val Lys Leu Ala Asp Phe Gly Se - #r Ala Ser Leu Val         Asp                                                                                              165  - #               170  - #               175              - - Pro Ala Gln Thr Phe Ile Gly Thr Pro Phe Ph - #e Met Ala Pro Glu Val                   180      - #           185      - #           190                   - - Ile Leu Ala Met Asp Glu Gly His Tyr Thr As - #p Arg Ala Asp Ile Trp               195          - #       200          - #       205                       - - Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Gl - #u Arg Arg Pro Pro Leu           210              - #   215              - #   220                           - - Phe Ser Met Asn Ala Met Ser Ala Leu Tyr Hi - #s Ile Ala Gln Asn Asp       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Pro Pro Thr Leu Ser Pro Ile Asp Thr Ser Gl - #u Gln Pro Glu Trp         Ser                                                                                              245  - #               250  - #               255              - - Leu Glu Phe Val Gln Phe Ile Asp Lys Cys Le - #u Arg Lys Pro Ala Glu                   260      - #           265      - #           270                   - - Glu Arg Met Ser Ala Glu                                                           275                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 273 amino - #acids                                                 (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                           - - Arg Glu Glu Arg Glu Arg Arg Lys Lys Gln Le - #u Tyr Ala Lys Leu Asn       1               5   - #                10  - #                15                - - Glu Ile Cys Ser Asp Gly Asp Pro Ser Thr Ly - #s Tyr Ala Asn Leu Val                   20      - #            25      - #            30                    - - Lys Ile Gly Gln Gly Ala Ser Gly Gly Val Ty - #r Thr Ala Tyr Glu Ile               35          - #        40          - #        45                        - - Gly Thr Asn Val Ser Val Ala Ile Lys Gln Me - #t Asn Leu Glu Lys Gln           50              - #    55              - #    60                            - - Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Le - #u Val Met Lys Gly Ser       65                  - #70                  - #75                  - #80         - - Lys His Pro Asn Ile Val Asn Phe Ile Asp Se - #r Tyr Val Leu Lys Gly                       85  - #                90  - #                95                - - Asp Leu Trp Val Ile Met Glu Tyr Met Glu Gl - #y Gly Ser Leu Thr Val                   100      - #           105      - #           110                   - - Asp Val Val Thr His Cys Ile Leu Thr Glu Gl - #y Gln Ile Gly Ala Val               115          - #       120          - #       125                       - - Cys Arg Glu Thr Leu Ser Gly Leu Glu Phe Le - #u His Ser Lys Gly Val           130              - #   135              - #   140                           - - Leu His Arg Asp Ile Lys Ser Asp Asn Ile Le - #u Leu Ser Met Glu Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asp Ile Lys Leu Thr Asp Phe Gly Phe Cys Al - #a Gln Ile Asn Glu         Leu                                                                                              165  - #               170  - #               175              - - Asn Leu Lys Arg Thr Thr Met Val Gly Thr Pr - #o Tyr Trp Met Ala Pro                   180      - #           185      - #           190                   - - Glu Val Val Ser Arg Lys Glu Tyr Gly Pro Ly - #s Val Asp Ile Trp Ser               195          - #       200          - #       205                       - - Leu Gly Ile Met Ile Ile Glu Met Ile Glu Gl - #y Glu Pro Pro Tyr Leu           210              - #   215              - #   220                           - - Asn Glu Thr Pro Leu Arg Ala Leu Tyr Leu Il - #e Ala Thr Asn Gly Thr       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Pro Lys Leu Lys Glu Pro Glu Asn Leu Ser Se - #r Ser Leu Lys Lys         Phe                                                                                              245  - #               250  - #               255              - - Leu Asp Trp Cys Leu Cys Cys Val Glu Pro Gl - #u Asp Arg Ala Ser Ala                   260      - #           265      - #           270                   - - Thr                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 24                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 31                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                           - - GACGCTGGAT CCAAAGATAC TGGNCAAGGG NGC       - #                  - #              33                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 3                                                                (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 6                                                                (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 13                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 16                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 19                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                           - - GGNGTNCCAG TTNGTNGCNA T           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 11                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 14                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 18                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                           - - AAAGGAAGCA NAGNCAGNAA CGGAAGAT         - #                  - #                  28                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 19                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: -                                                                (B) LOCATION: 22                                                               (D) OTHER INFORMATION: - #/note= "Where N is inosine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                           - - GACGCTGAAT TCACCTTCNG GNGCCATCCA         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                           - - Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Le - #u Gln Tyr Arg Lys Met       1               5   - #                10  - #                15                - - Lys Lys Leu Leu                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                           - - Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gl - #n Lys Arg Glu Tyr Lys       1               5   - #                10  - #                15                - - Leu Arg Lys                                                                - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                           - - Arg Glu Leu Arg Glu Leu Glu Gln Arg Val Se - #r Leu Arg Arg Ala Leu       1               5   - #                10  - #                15                - - Leu Glu Gln Lys                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino - #acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                           - - His Arg Asp Ile Lys Ala Gly Asn                                           1               5                                                             __________________________________________________________________________ 

What is claimed is:
 1. An isolated polypeptide comprising an amino acid sequence provided in SEQ ID NO:2, or a variant thereof that is at least 80% identical to SEQ ID NO:2 and that differs from SEQ ID NO:2 only in one or more amino acid substitutions, additions of terminal amino acid residues and/or deletions of terminal amino acid residues, wherein the ability of the variant to phosphorylate MEK3 is not substantially diminished.
 2. A polypeptide according to claim 1, wherein the polypeptide comprises a sequence that differs from a sequence recited in SEQ ID NO:2 only in conservative substitutions, such that the polypeptide is at least 90% identical to SEQ ID NO:2.
 3. A polypeptide according to claim 1, wherein the polypeptide comprises amino acids 1-416 of SEQ ID NO:2.
 4. An isolated polypeptide comprising an amino acid sequence provided in SEQ ID NO:2 modified such that aspartic acid residue 169 is replaced with alanine.
 5. An isolated polypeptide comprising an amino acid sequence provided in SEQ ID NO:4, or a variant thereof that is at least 80% identical to SEQ ID NO:4 and that differs from SEQ ID NO:4 only in one or more amino acid substitutions, additions of terminal amino acid residues and/or deletions of terminal amino acid residues, wherein the ability of the variant to phosphorylate MEK3 is not substantially diminished.
 6. A polypeptide according to claim 5, wherein the polypeptide comprises a sequence that differs from a sequence recited in SEQ ID NO:4 only in conservative substitutions, such that the polypeptide is at least 90% identical to SEQ ID NO:4.
 7. A polypeptide according to claim 5, which is constitutively active.
 8. A pharmaeutical composition, comprising:(a) a polypeptide according to any one of claims 1-7; and (b) a physiologically acceptable carrier. 